Apparatus and methods for correcting for variable velocity in microfluidic systems

ABSTRACT

Electrokinetic devices having a computer for correcting for electrokinetic effects are provided. Methods of correcting for electrokinetic effects by establishing the velocity of reactants and products in a reaction in electrokinetic microfluidic devices are also provided. These microfluidic devices can have substrates with channels, depressions, and/or wells for moving, mixing and monitoring precise amounts of analyte fluids.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.60/049,013 filed Jun. 9, 1997 entitled “APPARATUS AND METHODS FORCORRECTING FOR ELECTROKINETIC EFFECTS IN MICROFLUIDIC SYSTEMS” byKopf-Sill and Parce (Attorney docket no. 017646-00360) and U.S. Ser. No.60/076,468 filed Mar. 2, 1998 “HIGH THROUGHPUT SCREENING APPLICATIONS OFMICROFLUIDIC SYSTEMS” by Cohen et al. (Attorney docket number100/04000); the present application claims priority to each of theseapplications and incorporates each of the applications herein in theirentirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention provides microfluidic apparatus, methodsand integrated systems for the separation and analysis of reactioncomponents, fluid velocities, component velocities and reaction rates.Exemplary software is provided.

BACKGROUND OF THE INVENTION

[0003] There exists a need for assay methods and associated equipmentand devices that are capable of performing repeated, accurate assaysthat operate at very small volumes. U.S. Ser. No. 08/761,575 entitled“High Throughput Screening Assay Systems in Microscale Fluidic Devices”by Parce et al. (see also, U.S. Ser. No. 08/881,696) provides pioneeringtechnology related to microscale fluidic devices, includingelectrokinetic devices. The devices are generally suitable for assaysrelating to the interaction of biological and chemical species,including enzymes and substrates, ligands and ligand binders, receptorsand ligands, antibodies and antibody ligands, as well as many otherassays.

[0004] In the electrokinetic devices provided by Parce et al., anappropriate fluid is placed in a microchannel etched into a substratehaving functional groups present at the surface. The groups ionize whenthe surface is contacted with an aqueous solution. For example, wherethe surface of the channel includes hydroxyl functional groups at thesurface, protons can leave the surface of the channel and enter thefluid. Under such conditions, the surface possesses a net negativecharge, whereas the fluid will possess an excess of protons, or positivecharge, particularly localized near the interface between the channelsurface and the fluid. By applying an electric field along the length ofthe channel, cations will flow toward the negative electrode. Movementof the positively charged species in the fluid pulls the solvent withthem.

[0005] Improved methods and devices for monitoring reactions betweenchemical or biological species would be desirable. Electrokineticmicrofluidic devices and assays using such devices are particularlydesirable, due to the general adaptability of electrokinetic movement ofsmall volumes of fluids to high throughput assay systems. The presentinvention fulfills these and a variety of other needs.

SUMMARY OF THE INVENTION

[0006] It has now been discovered that accurate determination of thereaction rate of a reaction conducted in a microscale fluidic device isfacilitated by consideration of the velocity of the components in thereaction. In a microscale system in which the flux of reactants andreaction products is conserved, the velocity of at least one reactant orproduct is determined and the concentration of a reaction product ismeasured or calculated, facilitating determination of the reaction rate.

[0007] The concentration of products and reactants is typically measuredat a selected position on the microscale fluidic device, e.g.,spectrophotometrically, radioscopically, electrochemically, oroptically. Velocity rates are optionally determined by measuring thespeed of a component in a portion of the microscale fluidic device overtime, or are determined by consideration of the parameters influencingvelocity, e.g., the charge and mass of the component in an electricfield. As described herein, methods of determining velocities are alsoprovided in a constant flux state by indirect measurements, e.g., thevelocity of a reactant or product can be determined by measuring adifferent reactant or product. Thus, any or all reactants or productvelocities can be observed or determined. Velocity markers are alsooptionally used to approximate velocity. In one series of embodiments,electrokinetic devices and fluid injection schemes are described whichself-correct for velocity effects on fluids.

[0008] A variety of reactants and products are assessed by thesemethods, including ligand and ligand binders such as an antibody and anantibody ligand, a receptor and a receptor ligand, biotin and avidin,proteins and complementary binding proteins, carbohydrates andcarbohydrate binding moieties, nucleic acids, etc. Reactions which aremonitored are fluorogenic or non-fluorogenic. A variety of microscaleapparatus are adaptable to the methods such as microvalve and micropumparrangements, and particularly electrokinetic devices and the like.Multiple reactants and products are optionally assessed by serial orsimultaneous detection methods or a combination thereof.

[0009] In one preferred class of embodiments, the microscale fluidicdevice provides for electrokinetic movement of reactants and productsalong a microfluidic channel. An electrokinetic microfluidic device isprovided, having a microfluidic channel. An electric field is appliedalong the length of the microchannel, thereby causing charged speciessuch as reactants, solvent molecules and products to move along thelength of the channel due to electrophoretic flow, as well as byelectroosmotic flow of the solvent in the channel. A first reactioncomponent having a first charge mass ratio (CM₁) and a first velocity(U₁) is contacted to a second reaction component having a second chargemass ratio (CM₂) and a second velocity (U₂) in the microchannel, therebypermitting formation of a reaction product with a third charge massratio (CM_(p)) and a third velocity (U_(p)). Additional reactioncomponents and products are optionally provided and assessed forvelocities and concentrations. In one embodiment, a reactant can have avelocity of zero, e.g., because it is fixed to a substrate of thedetection apparatus. However, the more typical case is for flowingreactants, where all reactants and products are flowing in channels ofthe system. Typically, the product has a velocity different from one ormore reactants in the system.

[0010] Apparatus for practicing the methods of the invention areprovided. For example, a microfluidic detection apparatus fordetermining the rate of formation of a moving analyte on anelectrokinetic microfluidic substrate is provided. The apparatus has amicrofluidic substrate holder for receiving a microfluidic substrateduring operation of the apparatus, having a microfluidic substrateviewing region. An analyte detector such as a phototube, photodiode, acharge coupled device, a camera, a microscope, a spectrophotometer, orthe like is mounted proximal to the substrate viewing region to detectthe moving analyte in a portion of the substrate viewing region. Acomputer operably linked to the analyte detector is provided. Thecomputer determines the rate of formation of the analyte, correcting forthe effects of the motion of the analyte, e.g., by determining orcollating the velocities of one or more components and theconcentrations of one or more components and calculating the rate offormation of one or more components, correcting for the velocity of thecomponents. In preferred embodiments, the apparatus also includes anelectrokinetic fluid direction system for moving fluids in themicrofluidic substrate, such as one or more electrodes which fit intowells of the substrate, operably coupled to one or more electrical powersupply.

[0011] Electrokinetic microfluidic devices are also provided. Thedevices have a substrate or body with a top portion, a bottom portionand an interior portion. The interior portion has at least twointersecting channels, with at least one of the two intersectingchannels having at least one cross sectional dimension between about 0.1μm and 500 μm. The device has an electrokinetic fluid direction systemfor moving an analyte through at least one of the two intersectingchannels, a detection zone for detecting the analyte within at least oneof the two intersecting channels, when the analyte is in motion, and adata detection device for detecting the analyte in the detection zone. Adata analyzer which determines a rate of formation of the analyte inmotion, such as a computer, is operably connected to the microfluidicdevice, e.g., with cables to the data detection device, or by recordingdata on the data collection device and transporting the recorded data(e.g., on a computer-readable storage medium) to the computer.Typically, the computer has appropriate software for determiningreaction rates and other related information.

[0012] In one embodiment, at least two intersecting channels are etchedin a top surface of the bottom portion, with the top portion being fusedto the top surface of the bottom portion, thereby forming the interiorportion disposed between the top portion and the top surface of thebottom portion. When heat lamination of glass or polymeric surfaces isperformed, the glass or polymer fuses, typically with no seam existingbetween the top and bottom portion of the resulting microfluidic chip.In one preferred embodiment, the top portion of the device has aplurality of wells in fluid communication with the electrokinetic fluiddirection system comprising an electrode adapted to fit into at leasttwo of the plurality of wells. By applying an electric current with theelectrode, solvent and analyte molecules are moved through the channels.

BRIEF DESCRIPTION OF THE DRAWING

[0013]FIG. 1 is a schematic depiction of the basic concept of continuousflow non-fluorogenic binding assays on microchips showing changes inelectrophoretic mobility over time and distance, including signaloutput.

[0014]FIG. 2 is a graph providing model predictions of a non-fluorogenicbinding assay with large association constant K_(a).

[0015]FIG. 3 is a graph providing model predictions of the fluorescencesignal of a non-fluorogenic binding assay at three values of theassociation constant K_(a).

[0016] FIGS. 4A-4C provide a schematic of an integrated apparatus of theinvention and flowchart operations of software for data manipulation.

[0017]FIG. 5 is a schematic of an exemplar fluorescent assay apparatusof the invention.

[0018]FIG. 6 is a schematic representation of a fluorescent assay of theinvention.

[0019]FIG. 7 is a schematic of the channel and reagent well layout ofCaliper LabChip™ designated “7A.”

[0020]FIG. 8 is a mobility shift signal measured (solid curve) for thebinding reaction of B-T₁₀-FL and streptavidin under a continuous flowmode for injection times of 2.5 s, 5 s, 10 s, 15 s. The concentrationsfor B-T₁₀-F and streptavidin were 3.1 μm and 78 nM, respectively. Thedashed curve depicts model predictions.

[0021]FIG. 9 is a mobility shift signal measured (solid curve) for thecompetitive binding reaction of B-T₁₀-Fl and biotin with streptavidinfor a 12-s injection time in a continuous flow mode. The concentrationsfor B-T₁₀-F and streptavidin were 3.1 μM and 78 nM, respectively. Theconcentrations of biotin were 0, 0.78, 1.6, 2.3, and 3.1 μM. The dashedcurve depicts model predictions.

[0022]FIG. 10 is a plot of the fluorescence level versus the reciprocalof the total concentration of biotin-containing species.

[0023]FIG. 11 shows experimental data and model calculations of anon-fluorogenic PKA enzyme assay in a continuous flow mode.

[0024]FIG. 12 shows an example of the progression of a phosphatasereaction on the exemplar fluorogenic substrate dFMUP.

[0025]FIG. 13 is a fluorescence trace of the titration of substrate in amicrochip phosphatase assay.

[0026]FIG. 14 is a fluorescence trace of the titration of substrate in amicrochip phosphatase assay as a function of inhibitor concentration.

[0027]FIG. 15 is a Lineweaver Burke plot used to determine Km and Ki forthe phosphatase assay.

[0028]FIG. 16 is the third hour of an eight-hour experiment for acontinuous flow phosphatase assay on a microchip with enzyme inhibition.

[0029]FIG. 17 is the summary of the eight-hour phosphatase inhibitionexperiment showing continuous inhibition for the duration of the study.

[0030]FIG. 18 is a schematic of the exemplar protease reaction on amicrochip.

[0031]FIG. 19 is the raw data from an exemplar protease reaction on amicrochip as a function of increasing FRET substrate concentration.

[0032]FIG. 20 is a Lineweaver Burke plot for determination of Km for theprotease assay.

[0033]FIG. 21 is the third hour of a twelve-hour inhibition experimentfor a continuous flow protease assay on a microchip.

[0034]FIG. 22 is the raw data for the first 1000 seconds of each of thefirst nine hours of a protease reaction for the continuous flowinhibition assay.

[0035]FIG. 23 is a summary of the inhibition observed for the first ninehours of the protease assay on a microchip.

[0036]FIG. 24 is a schematic of an exemplar kinase reaction on amicrochip.

[0037]FIG. 25 is a schematic of microfluidic devices used in performingthe non-fluorogenic kinase assays described herein (the “28A” and “28B”LABCHIPS™).

[0038]FIG. 26 is the fluorescence data and a Lineweaver Burke plot forthe Km determination for PKA in a microchip.

[0039]FIG. 27 is a fluorescence trace for the PKA assay demonstratingthe mobility shift observed when enzyme is pulsed into a continuousstream of fluorescent substrate for various periods of time.

[0040]FIG. 28 is a fluorescence trace for the protease assaydemonstrating the concentration dependent mobility shift observed wheninhibitor is pulsed into a continuous stream of substrate and enzyme fortwo concentrations of inhibitor.

[0041]FIG. 29 is an extended time phosphatase assay (hour 3 of an 8 hourdata run) with no reagent replacement.

DEFINITIONS

[0042] Flux (“J”) is equal to the velocity of analyte molecules(generally referred to herein as “U”) times the concentration of theanalyte molecules (generally referred to as “C”) in a selectedmicrofluidic system. Flux is “conserved” in a microfluidic system, suchas a microchannel, when U times C is constant for a selected set ofanalyte molecules, such as reactants, products or both. For example, ina three component system, having a first reaction component with a massconcentration C₁ and a velocity U₁, a second reaction component withvelocity U₂ and concentration C₂, and a product, with velocity U_(p) andconcentration C_(p), flux is constant whenU_(1w)C_(1w)+U_(2w)C_(2w)+U_(pw)C_(pw)=U_(1z)C_(1z)+U_(2z)C_(2z)+U_(pz)C_(pz)where w is one point in the channel and z is a second point in thechannel. An alternative notation is[U₁C₁+U₂C₂+U_(p)C_(p)]_(w)=[U₁C₁+U₂C₂+U_(p)C_(p)]_(z). A more generalnotation that allows for multiple product (P) or reactant (R) speciesis:${\sum\limits_{h = 1}^{m}{C_{R_{h}}U_{h}}} = {\sum\limits_{i = 1}^{n}{C_{P_{i}}U_{i}}}$

[0043] where C is mass concentration (not molar concentration), m is thenumber of species before the reaction, and n is the number of speciesafter the reaction. Thus, the sum of the mass concentration times thevelocity of each of the species before a reaction is equal to the sum ofthe mass concentration times the velocity of each of the species after areaction. In the cases when the reaction yields no net change in thetotal number of molecules, the molar flux as well as the mass flux areconserved.

[0044] “Velocity” typically refers to the distance a selected componenttravels (l) divided by the time (t) required for the travel. In manyembodiments, the velocity under consideration is essentially constant,e.g., for the travel of reaction components along the length of amicrochannel under a constant rate of current in an electrokineticsystem. Although products of reactions typically change velocity as theyare made from, or by, reactants, the velocity change is often consideredto be instantaneous because the product reaches its terminal velocity inthe system in a very short period of time. Thus, the velocity of aproduct is essentially constant immediately following formation of theproduct. Where the velocity changes significantly over time, due, e.g.,to change of applied current in an electrokinetic system, or where achange from substrate to product results in a slow acceleration (ordeceleration) in the system, an “instantaneous velocity” equal to thechange in distance for a selected time (Δl/Δt) can be determined bygraphing distance against time and taking the tangent of the resultingfunction at a particular point in time.

[0045] A “microfluidic” channel is a channel (groove, depression, tube,etc.) which is adapted to handle small volumes of fluid. In a typicalembodiment, the channel is a tube having at least one subsection with across-sectional dimension of between about 0.1 μm and 500 μm;ordinarily, the channel is closed over a significant portion of itslength, having top, bottom and side surfaces.

[0046] As used herein, “electrokinetic material transport systems” or“electrokinetic devices” include systems which transport and directmaterials within an interconnected channel and/or chamber containingstructure, through the application of electrical fields to thematerials, thereby causing material movement through and among thechannel and/or chambers, i.e., cations will move toward the negativeelectrode, while anions will move toward the positive electrode. Suchelectrokinetic material transport and direction systems include thosesystems that rely upon the electrophoretic mobility of charged specieswithin the electric field applied to the structure. Such systems aremore particularly referred to as electrophoretic material transportsystems. Other electrokinetic material direction and transport systemsrely upon the electroosmotic flow of fluid and material within a channelor chamber structure which results from the application of an electricfield across such structures. In brief, when a fluid is placed into achannel which has a surface bearing charged functional groups, e.g.,hydroxyl groups in etched glass channels or glass microcapillaries,those groups can ionize. In the case of hydroxyl functional groups, thisionization, e.g., at neutral pH, results in the release of protons fromthe surface and into the fluid, creating a concentration of protons atnear the fluid/surface interface, or a positively charged sheathsurrounding the bulk fluid in the channel. Application of a voltagegradient across the length of the channel, will cause the proton sheathto move in the direction of the voltage drop, i.e., toward the negativeelectrode. The steady state velocity of this fluid movement is generallygiven by the equation:$v = \frac{{\varepsilon\xi}\quad E}{4{\pi\eta}}$

[0047] where v is the solvent velocity, ε is the dielectric constant ofthe fluid, ξ·is the zeta potential of the surface, E is the electricfield strength, and η is the solvent viscosity. The solvent velocity is,therefore, directly proportional to the surface potential. Use ofelectrokinetic transport to control material movement in interconnectedchannel structures was described in WO 96/04547 to Ramsey, which isincorporated by reference.

[0048] A “ligand” is a molecule which selectively binds or “hybridizes”to a “ligand binding partner”. Many examples of ligands and ligandbinding partners are known, including biotin and avidin or steptavidin,substantially complementary strands of nucleic acids, proteins andmolecules bound by proteins (including cell receptors and cognatereceptor binding molecules, antibodies and cognate antigens, etc.),proteins and “complementary proteins” (proteins which are specificallybound by other proteins, such as a cell receptor and a peptide whichspecifically binds the cell receptor), carbohydrates and carbohydratebinding molecules, engineered associating peptides and the like.

[0049] An “aqueous” solvent comprises primarily water, and optionallyfurther comprises other chemical species, depending on the intendedapplication, such as buffers, dyes, preservatives, or the like.

[0050] A “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues of naturalnucleotides that hybridize to nucleic acids in manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence optionally includes the complementarysequence thereof.

[0051] An “antibody” is a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (“antigen” or “antibodyligand”).

[0052] A “label” is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. A “label moiety” is the detectable portion of thecomposition, e.g., the fluorophore, radioactive element or the like.

DETAILED DESCRIPTION

[0053] In some assays it is useful to determine the concentrations ofproducts and related reaction rates for reactions in microfluidicdevices. In standard laboratory devices where products or reaction ratesare determined, such as cuvettes, or systems where reactants aredelivered to reaction chambers, the analysis of reaction rates isstraightforward, since all components of the reaction are maintained inone location. The reaction rate is related to the concentration ofreagents and the time between the mixing of reagents and detection ofthe product. It has now been discovered that this simple analysis is notapplicable to microfluidic systems in which reaction components andproducts have differing velocities through the channels of the device.Methods of determining the reaction kinetics in electrokinetic systemsare provided.

[0054] In the case of electrokinetic movement of chemicals, the velocityof different chemical species in a single flowing system is notnecessarily identical. Velocity for a particular component depends onthe charge of the particular species, the size of the species, thesolvent, and the like. For example, in a standard electrophoresis gel,analytes such as nucleic acids move through the matrix of the gel atdifferent rates, depending on the size of the molecule and the charge ofthe molecule. Large molecules move more slowly in the matrix of the gel.Highly charged molecules have a greater attraction for an oppositelycharged electrode than more modestly charged molecules, making morehighly charged molecules travel toward an oppositely charged electrodewith a higher velocity. These basic properties are understood, and formthe basis for purification and analysis of biological and chemicalmolecules. However, mixing of components in such standardelectrophoretic systems is not performed. No attempt is made duringstandard electrophoresis to determine reaction rates for the mixing ofreactants. Accordingly, the special problems encountered duringelectrokinetic mixing were not considered in the electrophoretic art,and, of course, solutions to these unknown problems were not proposed.

[0055] In the special case of electrokinetic movement of fluids in amicrofluidic device, different species are commonly mixed to form one ormore product. Any or all of the reactant species or reaction productscan have differing mobilities. Thus, for example, an enzyme can bereacted with a substrate which is modified to form a product. Thesubstrate, modified substrate (i.e., product) and enzyme will often allhave different mobilities. Detection equipment downstream from areaction site in the microfluidic device will perceive the concentrationof reactants and products based, in part, on the differing velocities ofthe components. For example, if an enzyme and a substrate are mixed atthe start of a microchannel down which the components travel, theappearance of any product of the reaction downstream to the reactionsite will depend on standard considerations such as the actual rate ofthe reaction (i.e., the number of product molecules made per unit timein the reaction), and the concentration of the reactants (until non-ratelimiting amounts of reactants are provided, the more reactants provided,the faster the reaction will proceed—a simple result of chemicalequilibrium). However, the perceived concentration of product downstreamof the reaction site also depends on the velocity of the product. Forexample, if the velocity of the product is substantially slower than thevelocity of the substrate in the system, then the product concentrationwill be substantially higher than the decrease in the substrateconcentration that produced it. This is in contrast to the standardnon-flowing system in which product concentration would be equal to thesubstrate that produced it. Thus, the reaction rates determined withoutconsideration of velocities of the system components were discovered notto match results for reactions obtained by standard techniques, wherethe velocity of the components is zero (or at least not changing).Accordingly, the present invention relates to the discovery of a problemnot previously known to exist, and to non-obvious solutions to this newproblem.

[0056] Although the analysis of reaction rates in an electrokineticsystem requires corrections for velocity changes, the value ofdetermining reaction rates for many different concentrations in veryshort periods of time and in very small volumes of fluids makes theeffort worthwhile. Accordingly, the present invention makes possible,for the first time, the accurate and simple analysis of accuratereaction kinetics in an electrokinetic system. The ability to assessreaction kinetics “on the fly” Le., with the reaction occurring whilethe components have velocity relative to the observer, greatly speedsthe rate at which such reactions can be assessed. This, in turnfacilitates accurate high-throughout determination of reaction kinetics,and of a variety of other flowing interactions with applicability todrug screening, nucleic acid sequencing, enzyme kinetics, and the like.

[0057] Uses for Correcting for Electrokinetic Effects

[0058] It will be appreciated that the ability to quickly and accuratelymonitor and determine reaction kinetics has broad applicability to manydifferent combinatorial approaches in biology and chemistry, for medicaldiagnostics, basic research, quality control, and the like. For example,the ability to correct for electrokinetic effects in microfluidicelectrokinetic systems enhances the versatility of such systems. Any andall uses contemplated for electrokinetic systems can benefit from thepresent methods of correcting for electrokinetic effects.

[0059] The present methods and compositions are useful in measuring therate of essentially any chemical or biological reaction, includingparticularly those which occur in an aqueous or other flowable solution.The methods are particularly desirable where repetitive screening ofreactants is needed. This has general applicability to assessing thepurity and activity of industrial and laboratory reagents (See, e.g.,Kirk-Othmer Encyclopedia of Chemical Technology third and fourtheditions, Martin Grayson, Executive Editor, Wiley-Interscience, JohnWiley and Sons, NY, and in the references cited therein (“Kirk-Othmer”)for a basic discussion of industrial chemical processes). Combinatorialscreening of large libraries of compounds for biological activitiesprovides the basis for finding new therapeutics. Thus, the ability tomonitor the effect of compounds on biologically relevant reaction ratesis of great importance and is of immediate commercial value to a varietyof pharmaceutical, agricultural and chemical industries.

[0060] Similarly, the ability to rapidly and accurately screen largepatient populations for evidence of infection, genetic disease, or thelike, is typically performed by monitoring the interaction of chemicalor biological components. For example, binding of HIV antigens toantibodies in a patient's blood is commonly used to detect whether apatient has been exposed to HIV. In a system in which the bindingconstant between the antibody and the relevant antigen can easily bemonitored, it is possible to reduce the incidence of false-positives.Thus, the present invention provides for increased sensitivity inbiological assays, as well as increased throughput.

[0061] In addition to monitoring antibody-antigen and otherprotein-protein interactions, it is possible to monitor the affinity ofnucleic acid-nucleic acid interactions. This is particularly useful forempirically determining percent similarity for complementary relatednucleic acids, and for detecting nucleic acids in various biologicalsamples (including PCR samples; See, PCR Protocols A Guide to Methodsand Applications (Innis et al. eds) Academic Press Inc. San Diego,Calif. (1990) (Innis)). As an alternative to standard solid stateSouthern or northern analysis (See, Sambrook, Ausubel, or Berger,supra.) the assay provides increased automation, a clear indication ofthe efficiency of nucleic acid hybridization (providing an increase insignal to noise ratios) and the like.

[0062] Monitoring reaction rates between enzymes and substrates hasapplicability as a general laboratory tool for basic research, where thereaction rate is unknown, and as a quality control tool for theassessment of the quality of reagents such as enzymes or substrates. Andin diagnostic assays. Enzymes and other chemical and biologicalcatalysts are in common use as components of foods, food supplements,detergents, therapeutics, and, e.g., as laboratory tools for recombinantnucleic acid manipulation (e.g., restriction enzymes, see, Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook);and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel) for adiscussion of some enzymes commonly used in molecular biology).Defective enzymes also serve as the direct cause for the etiology ofmany inherited diseases, including, e.g., ADA and phenylketonuria. Theability to screen enzymes rapidly from patients suffering enzyme defectsis of considerable medical diagnostic value.

[0063] Methods of Correcting for Electrophoretic Effects

[0064] The present invention provides methods of accurately determiningthe rate of a chemical reaction. The reaction can be between two or morecomponents that chemically join (by forming a covalent or non-covalentassociation) to form a new component or complex, or between a componentsuch as an enzyme, catalyst or electromagnetic radiation that converts afirst reactant or other component into a product, or due to spontaneousdegradation of a component. In the methods, a first component and asecond component are contacted, often by mixing, typically in a channelin an electrokinetic device. The components react to form a product.

[0065] Flux (J), with units of molecules/(cross sectional area×time) ormass/cross sectional area×time, is equal to the velocity of themolecules under consideration (U) times the concentration of molecules(C); thus, J=U×C. Flux is conserved in the microchannel. In other words,the number of analyte molecules (enzymes, substrates and products, orligands and ligand partners) times the velocity of the components in amicrochannel is constant along the channel.

[0066] The components and the solvent all travel along the length of thechannel at different velocities to a position downstream of the mixingpoint where they are detected, typically by detecting a label (a varietyof labels are described supra).

[0067] The velocity of one or more reaction components (U_(r1), U_(r2),U_(r3) . . . ) or products (U_(p1), U_(p2), U_(p3) . . . ) in thechannel are determined. As explained in the examples below, in a systemin which flux is conserved, if the velocity of one component is known,the velocities of the other components can be determined, givenconcentration information, charge mass ratios (ordinarily, the chargemass ratio (CM) is proportional to velocity in a flowing system, i.e.,U_(r1) is proportional to CM_(r1), U_(r2) is proportional to CM_(r2), U₃is proportional to CM_(r3), U_(p1) is proportional to CM_(p1) . . . .),or the like. In some unusual instances, velocity (U) and charge massratios (CM) are not directly proportional due to unusual molecularshapes which either shield charge on portions of the molecules, or whichcause molecular drag during electrophoretic motion.

[0068] In one convenient embodiment, the velocities of the reactants areknown, either from direct measurement, or from previous measurements ina similar system, or by comparison to known velocity markers. Velocitymarkers are components which are run in the system which are detectableand known to have a particular velocity relative to an analyte.Measurement of the marker is used to estimate the velocity of theanalyte (reactant, product or the like). The product velocity may besimilarly known, or directly measured, e.g., by measuring the velocityof a detectable product over a section of the microchannel. Similarly,the velocities of the reactants can be measured over a section of themicrochannel.

[0069] The concentration of the reaction product is determined in) aportion of the microchannel. This determination can be done by measuringthe number of molecules with the detector as described above, typicallyin a given section of an electrokinetic channel. Alternatively, theconcentration can be determined indirectly, by measuring velocities andconcentrations of other components in the system. Where flux isconserved, the sum of the concentration of reactants and products timesthe respective velocity of reactants and products is constant.Accordingly, the concentration of particular components can be measured,or determined from measurements for other components in the system,e.g., using simple algebra. For example, in a simple system havingreactant 1 (R1) reactant 2 (R2) and a product (P) where J is constant,and J=(U_(R1))[R1]+(U_(r2))[R2]+(U_(p))C_(p), one of skill can easilydetermine C_(P) where J is constant andJ=[U₁C₁+U₂C₂+U_(P)C_(P)]_(w)=[U₁C₁+U₂C₂+U_(P)C_(P)]_(z). By algebraicmanipulation,C_(Pz)=(U₁/U_(p))(C_(1w)−C_(1z))+(U₂/U_(p))(C_(2w)−C_(2z))+C_(Pw).Similar algebraic considerations can be used to yield the velocities orconcentrations of other components where sufficient information isavailable. Linear algebra techniques are conveniently used to solve forthe concentrations or velocities of components where there are multipleunknowns related in multiple flux relationships.

[0070] Given the velocity of a product (U_(p)) and the concentration ofa product (C_(p)), it is possible to correctly determine the rate of areaction. In particular, it is possible to determine the rate at which aproduct is formed, by conversion of one or more of the reactants into aproduct.

[0071] In the system in which one of the reactants aids in convertingthe other reactant into the product (e.g., where R1 is an enzyme orcatalyst and R2 is a substrate), the following flux relationship can beused in determining a reaction rate: Flux(3)=[R1]×T_(LR2)×k×U_(R2)=[R2]_(converted)×U_(p)=U_(p)×C_(P), where k isthe turnover number for the enzyme reaction. Rearranging and writingtransit time (T_(LR2)) of substrate as L/U_(R2) results in:[R1]×L/U_(R2)×k×U_(R2)=U_(p)×C_(P). Thus, [R1]/U_(p)×L×k=C_(p).Substituting transit time for product (_(TLP)) for L/U_(p) gives theresult that product concentration is proportional to the transit time ofthe product, not the substrate as might have been extrapolated from thestationary or non-mobility changing case above: [R1]×T_(LP)×k=C_(P).Thus, k=C_(P)/([R1]T_(LP)). In one embodiment, where the productconcentration before a reaction is zero and the enzyme concentration, R1remains essentially constant, then, rearranging,C_(P)=([R2]_(total)−[R2]_(unreacted))U₂/U_(p).

[0072] Consideration of the case in which two or more components arejoined to form a product is similar. When two reactants join, theytypically result in a product with a different velocity than either ofthe two individual reactants (R1 and R2). With the flux being conserved,the concentration of detected species changes as a result of a change invelocity. The product optionally results in a different detectable labelthan either of the reactants, or can have the same label. Where R1 andR2 molecules are converted to P, taking the principle of theconservation of flux into account:

[R1]×U _(R1) =C _(P) ×U _(P)

[0073] Recognition of this relationship allows quantification of theamount of R2 present in the system by detecting downstream fluorescence(all R2 is bound to R1). The relationship between the concentrations ofR1 bound to R2 (i.e., forming P) and unbound R1 is proportional to theirmobilities: C_(P)=[R1]×U_(R1)/U_(P).

[0074] At intermediate amounts of R2, where a portion of R1 is bound toR2, the concentration is proportional to the fraction (Y_(R1)) of R1that is bound to R2:

C _(P) =Y _(R1)([R1]U _(R1) /U _(p)).

[0075] Without the knowledge that concentration changes as velocitychanges, as taught herein, the assay is necessarily more complicated.For example, one could sample the mixture into a separation column whichseparated reacted and unreacted molecules, and detected florescence. Theamount of material coming off of the column per unit time could bedetected (see also, the Examples below). However, using conservation offlux, much simpler arrangements are possible. For instance, anelectrokinetic system with one channel and two electrodes driving fluidflow in an electrokinetic device is used to monitor formation ofreaction products.

[0076] It will be appreciated that products and reactants need not befluorogenic (producing or quenching a fluorescent signal), but only needto be “velocitigeneic,” i.e., a reaction need only produce a detectablechange in velocity of a product compared to a substrate. This ability tosort signals based on the velocity of products as compared to reactantsprovides for the detection of multiple reactions and multiple productsin a single electrokinetic device. Additional assays utilizingnon-fluorogenic assays are described below.

[0077] A mass balance on the substrate of an enzyme reaction yields:

[S] _(total) =a[S] _(converted)+(1−a)[S] _(remaining),

[0078] where “a” is the fraction of substrate (S) that is converted toproduct. By definition, [S]_(converted)=C_(P).

[0079] From the conservation of flux: C_(P)=[S]×U_(s)/U_(P). Therefore,[S]=a[S]U_(s)/U_(p)+(1−a)[S]. After measuring the signal before thereaction (l.h.s.) and after the reaction (τ.h.s.), it is possible tosolve for “a” if the velocity of substrate and product, U_(s) and U_(P),are known.

[0080] In many enzyme reactions, enzyme kinetics are studied in a rangein which a very small portion of substrate is converted into product; inthese cases, the substrate concentration can be treated as a constant.This makes the signal change due to formation of the product relativelysmall. To optimize the signal to noise ratio for observation of theproduct, it is possible to optimize electrokinetic flow so that theproduct velocity is slow (or close to zero) when the substrate velocityis relatively high, or to make product velocity fast while substratemobility is slow.

[0081] When reactions are performed on microsubstrates withelectrokinetic movement of solutions, the analysis of reaction rates andproduct formation is done from a starting point of conservation of flux.This is in contradistinction from prior art systems in which thevelocities of reactants and products do not differ, permitting analysisfrom a simple standpoint of concentration balance. The presentinvention, therefore, provides for correct determination of reactionrates, a wider range of detectable reagents (e.g., velocitigenic, ratherthan flourescent), and simpler electrokinetic movement and detectionapparatus.

[0082] Non-Fluorogenic Assays

[0083] The detection of results for many biochemical assays inconventional cuvette experiments, as well as in microfluidic devices hasprimarily been based on fluorogenic or chromogenic reactions in whichthe quantum efficiency of a labeling fluorescent moiety or the amount ofcolored label (chromophore) changes as a result of the reaction.However, for certain classes of assays the reactions are non-fluorogenic(i.e., there is no change in the quantum efficiency of the labelingfluorescent species upon reaction by the enzyme). As noted above, areaction need only be velocitigenic for accurate rate determination; theformation of a new detectable element is not necessary in the practiceof the invention.

[0084] It will be appreciated that the concepts described fornon-fluorogenic assays are equally applicable for non-fluorescentsystems, in which the label is other than a fluorophore, i.e., acalorimetric label, a radioactive label, an electrochemical label, orthe like; for example, a non-chromogenic assay is an assay in which thecolor or intensity of a label does not change upon reaction; anon-radiogenic assay is an assay in which the radioactive component ofthe label is not modified by the reaction. Again, the relevant criterionis that a product have a different velocity than a reactant. Forsimplicity, fluorogenic assays and non-fluorogenic assays are discussedin more detail; it will be appreciated upon review of this disclosurethat similar considerations apply for radio labels, chromophore labels,pH labels, ionic labels, or other common labels known to one of skill.

[0085] Detection of non-fluorogenic assays is possible in anelectroosmotically driven microfluidic device using periodic injectionsof reaction mixture into a separation channel, in which reactants andproducts are separated by electrophoresis due to changes in theelectrophoretic mobility resulting from the reaction, as discussed above(see also, A. R. Kopf-Sill, T. Nikiforov, L. Bousse, R. Nagel, & J. W.Parce, “Complexity and performance of on-chip biochemical assays,” inProceedings of Micro- and Nanofabricated Electro-Optical MechanicalSystems for Biomedical and Environmental Applications, SPIE, Vol. 2978,San Jose, Calif., February 1997, p. 172-179). The periodic injectionsare typically on the order of from about 0.0001 to 10 minutes, typicallyabout 0.001 to 1 minute, often about 0.1 seconds to 10 second. See also,concurrently filed U.S. application Ser. No. ______ (attorney docketnumber 100/04200).

[0086] In an alternate non-fluorogenic continuous flow mode assays ofthe invention, the injection/separation step is eliminated. The bindingreaction of fluorescently-labeled biotin to streptavidin was chosen as amodel system for non-fluorogenic continuous flow mode.

[0087] The following discussion provides the basic concept of continuousflow non-fluorogenic assay on microchips, the use of conservation offlux to predict and interpret non-fluorogenic assay data quantitatively,modeling and experimental information to validate these concepts,applications of the format to biochemical assays on microchips, and theapplicability of non-fluorogenic assays e.g., to high throughput drugscreening.

[0088] The Continuous Flow Non-Fluorogenic Assay Format

[0089] In an electroosmotically driven microfluidic device, each type ofdissolved species in a buffer moves down a channel at a velocity(U_(tot)) equal to the vector sum of the electroosmotic velocity of thebuffer (U_(eo)) and the electrophoretic velocity of the molecule(U_(ep)):

U_(tot)=U_(eo)+U_(ep)=(μ_(eo)±μ_(ep))E.

[0090] In this equation, μ_(eo) and μ_(ep) are the electroosmoticmobility of the buffer and the electrophoretic mobility of the dissolvedspecies, respectively, and E is the applied electric field. Theelectrophoretic mobility in turn depends on the charge-to-mass ratio ofthe molecule. In most biochemical reactions, the charge-to-mass ratio ofthe reactant molecule changes as a result of the reaction, thus changingthe electrophoretic mobility of the molecules. This change in mobility,and therefore velocity, is the basis for detection of non-fluorogenicreactions in a continuous flow format.

[0091] Accordingly, methods of determining concentration of a reactionor assay product (C_(p)) in a channel of a microfluidic device areprovided. In the methods, a labeled first reactant or assay componenthaving a velocity (U_(r)) and a label (L_(r)), such as a fluorophore,chromophore or other label (see, supra for a discussion of labels) isflowed down a microfluidic channel and past a signal detector (detectorsare also described supra). The labeled first reactant or assay componentproduces a signal (S_(as)) detectable by the detector. The labeled firstreactant or assay component is converted to a reaction or assay productcomprising a label L_(p), the product having a velocity (U_(p)). In thetypical case, (U_(r)) does not equal (U_(p)), resulting in a change insignal from L_(p), thereby providing an indication of C_(p). Because theassay is non-fluorogenic, L_(p) comprises component elements of L_(r)(i.e., the labels are typically essentially the same for the product andreactant, i.e., providing the same detectable output). Reactant or assaycomponent signal (S_(as) of a labeled first reactant or component priorto addition of a second reactive component, termed “S_(r)”) can besubtracted from S_(as) after the addition of additional components whichreact with the first reactant or component to provide a normalizedsignal (S_(n)) produced by the product.

[0092] In non-fluorogenic assays, a molecule comprising L_(p) isconverted from a molecule comprising L_(r) by treating the molecule withany physical component or force which results in a modification of themolecule, including light, heat, electrical charge, a polymerizationagent, a catalyst, or a binding molecule. L_(r) and L_(P) are optionallyidentical after the conversion, with only distal portions of themolecule being affected. Alternatively, L_(r) can be modified so that anew label, L_(p), is produced; however, the output of the labeltypically does not change in a non-fluorogenic assay. Of course, wherethe label does change, the concepts herein can also be applied, as thevelocity will typically also concomitantly change.

[0093] The basic concept of the continuous flow mode of anon-fluorogenic assay can easily be illustrated with a schematic drawingof a binding reaction as shown in FIG. 1. In FIG. 1, thefluorescently-labeled reactant molecules are denoted by circles and theunlabeled reactant are denoted by squares. The reaction productmolecules, denoted by the combined shape of a circle and a square, areshown lighter toned as a result of a binding reaction which, for thesake of simplifying this discussion, is fast and has a high associationconstant (K_(a)). (K_(a)=[P]/[A][B] for a reaction A+B→P, where thebrackets denote concentrations.) The labeled reactant (circles) flowscontinuously down the main channel at a constant concentration, whereasthe unlabeled reactant (squares) is injected in a short pulse from aside channel into the main channel. In this illustration, the labeledreactant is assumed to move slow whereas the product moves fast (in thefigure, motion is from left to right).

[0094] As the squares are injected into the main channel, they bind tothe circles and convert them to fast moving molecules (for purposes ofsimplification, the binding is considered to be instantaneous).Downstream of the injection point, the faster moving product catches upwith the slower reactant, giving rise to a higher local concentration offluorescent species (i.e., the sum of labeled reactants and labeledproducts) ahead of the injection plug, and a lower concentration at thetrailing end of the injection plug due to the depletion in reactants.Quantitatively, it is important to recognize that the product zoneoccupies a larger volume in the channel than the depleted reactant zonedue to the higher product velocity. Consequently, the apparentconcentration of product in the channel is less than the concentrationof the reacted reactant, since the same number of molecules are nowspread out in a larger volume. Interestingly, in the time domain asillustrated in the bottom of FIG. 1, the widths of the peak and valleyare the same because the spatially wider product zone, which has beenincreased by a factor equal to the ratio of product velocity (U_(p)) toreactant velocity (U_(r)), moves past the detector faster by the samefactor of U_(p)/U_(r). If the concentration of the reacted reactant(C_(p)) and the velocities U_(r) and U_(p) are known, the concentrationof the product (C_(p)) can be calculated as: C_(p)=C_(r)(U_(r)/U_(p)).This equation makes use of the concept of conservation of flux (flux isdefined as the product of velocity and concentration as discussedabove).

[0095] When a label detector is placed downstream of the injection point(e.g., a photomultiplier tube, photo diode, or the like), depending onthe distance between the injection and detection points, the length ofthe injection plug, and the species velocities, the plug of fastermoving product can be partially or totally separated from the slowermoving depletion hole of the reactant. In the case of partialseparation, the detector signal (S_(as)) displayed in time will show acharacteristic shape of a peak followed by a plateau region and avalley. The ratio of the magnitude of the peak to valley is(C_(p)/C_(r)), which, by algebraic manipulation, is equal to(U_(r)/U_(p)). The plateau region is lower in fluorescence than thebackground level. The ratio of the magnitude of the plateau region tothe valley is 1−(C_(p)/C_(r)) or 1−(U_(r)/U_(p)). In the case of totalseparation, the signal shows a peak and a valley separated by thebaseline fluorescence level instead of the plateau region.

[0096] Mobility Shift Modeling

[0097] For the case of a fast binding assay with a high K_(a) (e.g.,between about 10⁵ and 10¹⁵ or higher, typically higher than about 10⁸M⁻¹for a 1 μM concentration of reactants) as described in the last section,the fluorescence signal can easily be modeled in the time domain, e.g.,using an Excel™ spreadsheet. Input parameters include reactantconcentrations, electroosmotic mobility of the buffer, electrophoreticmobilities of the labeled reactant and product, distance betweeninjection and detector locations, injection pulse time, and appliedfield strength. See, Appendix 1.

[0098] Two cases of the model predictions are shown in FIG. 2. The firstcase, denoted by the solid curve in FIG. 2, is for a long injection timesuch that the signal peak and valley are only partially separated and aplateau region is clearly seen. The second case (dash curve) is for ashort injection time such that the peak and valley are fully separatedby the baseline fluorescence level. Note that in both cases, themagnitude of the peak height is smaller than the magnitude of the valleydepth due to the principle of conservation of flux in flowing systems.

[0099] For the more general case of a reaction with variable reactionrates and K_(a) values, continuous flow non-fluorogenic assays can bemodeled in the spatial domain. In one convenient embodiment, an Excelspreadsheet is again utilized. The basic construct of the spatial domainmodel is to split the channel into discrete sections spatially and intime. At an initial time, the channel is filled with the labeledreactant. For each subsequent time step, the second reactant is allowedto be injected into the channel and then reacted with the labeledreactant to form products at some prescribed reaction kinetics, whichare required as input parameters. In this model, an algorithm isincluded to ensure that the concentration flux of each species movingdown the channel is conserved. The Macro program listing, in VisualBasic Applications (VBA), for binding assays with variable K_(a) valuesis included in Appendix A. FIG. 3 illustrates model predictions of thefluorescence signal at various values of K_(a) when the concentrationsof the reactants were chosen to be 1 μM.

[0100] Integrating Non-Fluorogenic Assays in High-Low Salt Format

[0101] In one series of high throughput screening embodiments, compoundsof interest (e.g., potential drugs, or other analytes) are dissolved ina high salt buffer and placed in a source of materials, such as thewells of a microtiter dish, with a low salt buffer used as the runningbuffer to pipette the compounds from the wells into the planar LabChip™,e.g., through a capillary. A variety of source-chip arrangements andinterfaces are described in 08/835,101 and CIP application 09/054,962 byKnapp et al. See also, U.S. Ser. No. 08/671,986. In brief, anelectropipettor pipettor having one or several separate channels isfluidly connected to an assay portion of the microfluidic device (i.e.,a microfluidic substrate having the reaction and/or analysis and/orseparation channels, wells or the like). In one typical embodiment, theelectropipettor has a tip fluidly connected to a channel underelectroosmotic control. The tip optionally includes features to assistin sample transfer, such as a recessed region to aid in dissolvingsamples. Fluid can be forced into or out of the channel, and thus thetip, depending on the application of current to the channel. Generally,electropipettors utilize electrokinetic or “electroosmotic” materialtransport as described herein, to alternately sample a number of testcompounds, or “subject materials,” and spacer compounds. The pipettorthen typically delivers individual, physically isolated, sample or testcompound volumes in subject material regions, in series, into the samplechannel for subsequent manipulation within the device. Individualsamples are typically separated by a spacer region of low ionic strengthspacer fluid. These low ionic strength spacer regions have highervoltage drop over their length than do the higher ionic strength subjectmaterial or test compound regions, thereby driving the electrokineticpumping, and preventing electrophoretic bias. On either side of the testcompound or subject material region, which is typically in higher ionicstrength solution, are fluid regions referred to as first spacer regions(also referred to as high salt regions or “guard bands”), that contactthe interface of the subject material regions. These first spacerregions typically comprise a high ionic strength buffer solution toprevent migration of the sample elements into the lower ionic strengthfluid regions, or second spacer region, which would result inelectrophoretic bias. The use of such first and second spacer regions isdescribed in greater detail in U.S. patent application Ser. No.08/671,986. These electropipettors are used to physically sample asource of materials of interest, such as a microtiter dish, a membranehaving dried or wet samples disposed thereon (dry samples can beresolublized, e.g. by expelling fluids from the electropipettor followedby drawing the expelled fluid into the device; for other arrangementssee 09/054,962) or the like.

[0102] In the high-low salt format, the electric field within the highsalt region in the channel of a pipettor chip is relatively smallcompared to that in the low salt region, due to the lower electricalresistance of the high salt buffer. Consequently, electrophoresis ofcompounds in the high salt plug is greatly retarded, whereas the highsalt plug itself is dragged along by electroosmosis driven primarily bythe conditions in the low salt region.

[0103] At least two general approaches to integrate non-fluorogenicassays into this high-low salt pipettor chip format for high throughputdrug screening--continuous flow mode and injection/separation mode areprovided. In the continuous flow format, integration of the two opposingprinciples of preventing and encouraging electrophoresis at will intoone simple chip design requires careful chip and experimental design.One method is to inject a buffer into the latter part of the mainreaction channel to “spoil” the high-low salt format after the assay hashad adequate incubation time to generate product.

[0104] Incorporating non-fluorogenic assays into the high-low saltformat by injection followed by separation in another channel is likelyto be less dependent on the buffer systems, and thus is general in itsapplicability to a wide range of biochemical assays. However, a controlmechanism is used to time the injection. External control mechanisms totime the arrival of the high salt plug to trigger injection include useof an electromagnetic means such as an in-situ conductivity probe in thechannel and/or optical methods based upon the intrinsic properties ofthe buffer (e.g., refractive index changes in high/low salt buffers), orplacing a dye marker in the buffer in conjunction with using an opticaldetector to time the flow. Another method is to use the pressuredeveloped at the interfaces of the high and low salt regions to induceinjection at a channel intersection. In this case, the injection isautomatic; no external control and feedback means is required. See also,concurrently filed U.S. application Ser. No. ______ (attorney docketnumber 100/04200).

[0105] Continuous Flow Assay Formats Using Interference Patterns ofAnalyte

[0106] Concentration Waves in Electrokinetic Microfluidic Systems

[0107] Methods to enhance the detection of non-fluorogenic assays onchips for small mobility shifts are available. One approach is to injectthe reaction mixture into a planar cyclic capillary electrophoresischannel to separate products from reactants. In this case, theseparation time can be made very long by continuously cycling thevoltage around the cyclic structure. Another method is to use theconcept of interference of concentration waves in channels to enhance tothe magnitude of peaks and valleys in the non-fluorogenic assayfluorescence signal (see, below).

[0108] Use of Concentration Waves for Data Correction

[0109] In a microfluidic device in which an electric field is appliedalong the length of the microchannel, charged species such as analytes,solvent molecules, reactants and products move along the microchannel bythe electrokinetic forces of electroosmosis and electrophoresis. The netmobility of each species is determined by the vectorial sum of theelectroosmotic and electrophoretic mobilities, the latter of which is afunction of the hydrodynamic radius-to-charge ratio of each species.During a chemical or biological reaction such as ligand-receptorbinding, antibody-antigen binding, etc., the reactants in general havedifferent electrophoretic mobilities than those of the products. Thedifferences in mobilities are useful for detection, e.g., ofnon-fluorogenic assays described above, in which reaction detection isnot dependent on the production or quenching of fluorescence as aconsequence of the reaction. Instead, the mobility difference duringflow in the microchannel is used to separate the “reactant hole” (i.e.,decrease in reactant concentration) of the labeled reactant from the“product peak” (i.e., increase in product concentration) undercontinuous flow, thereby providing a signature from which quantitativeinformation on the reaction kinetics can be extracted from calculationmethods based on conservation of species flux discussed supra.Non-fluorogenic assay formats are unique to electrokinetic microfluidicsystems; there is no analogy for cuvette assays.

[0110] The invention provides methods for performing continuous flowassays in electrokinetic microfluidic devices to facilitatedetermination of reaction kinetics using the generation and detection ofreactant and product “concentration waves” in microchannels. Thereactant concentration wave is generated temporally by modulating theconcentration of one or more reactants using electroosmotic pumping. Theproduct/concentration wave is generated as a result of the reaction. Atthe point of reaction in the microchannel, the product wave isinherently 180° out-of-phase with the reactant wave. If the reaction isnon-fluorogenic, a detection device placed very close to the point ofreaction along the microchannel measures a constant signal (such as dueto fluorescence of a labeling moiety covalently bonded to a reactant),since the sum of the signals from the labeled reactant and convertedproduct is constant. Further downstream of the microchannel, however,the reactant and product waves separate spatially due to differences inelectrophoretic mobility, and the reaction can be detected. The measuredsignal can be viewed as “interference” of the reactant and productwaves, analogous to the phenomenon of interference of electromagnetic(such as optical) waves. The “phase shift” in the reactant and productwaves is a function of the net mobility difference of the labeledreactant and product, the average flow velocity in the microchannel, andthe distance from the point of reaction. At the point of reaction, thephase shift is zero and the waves interfere destructively. As the phaseshift approaches 1800, the waves interfere constructively and the signalis maximized.

[0111] In studying the kinetics of a reaction in a microfluidic device,analyte concentration waves with a constant frequency and varyingconcentrations can be used to elucidate the dependence of kinetics onconcentration (analogous to analyte titration). An “interferencepattern” as a function of spatial position can be measured by placingthe detector at different points along the microchannel. Deconvolvingthe interference patterns using wave equations, conservation of flux,and diffusion equations provides quantitative information on speciesmobilities and reaction kinetics.

[0112] In many cases when the mobility shift of the reactant and productis not known, a reactant concentration wave with varying frequencies canconveniently be used to study the reaction. For instance, the frequencyof the reactant concentration wave can be increased linearly with time.A detector located at a fixed distance from the point of reaction canmeasure an increase in the signal intensity as the mobility-inducedphase shift becomes a significant fraction of the wavelength of theconcentration wave. Again, kinetics data can be obtained by deconvolvingthe signal using wave, diffusion, and flux conservation equations.

[0113] In general, this continuous flow assay format using interferencepatterns of analyte concentration waves can be applied to a wide rangeof assays. This format can be especially sensitive to small changes inthe mobility shift of the converted product, such as in the case ofligand-receptor assay, in which the mobility of the protein-ligandcomplex is expected to differ little from that of the labeled protein ofinterest since the binding ligands are usually small molecules. Thefollowing is an example to illustrate the practicality and usefulness ofthis format. The reaction of interest is:

P+L→PL

[0114] where P is a labeled protein with molecular weight of 10 to 100kDaltons, L is a ligand with molecular weight of 50 to 500, and PL isthe protein-ligand complex. If a concentration wave of an unlabeledligand is electroosmotically pumped into a microchannel containing aconstant concentration of the labeled protein, the binding reactiongenerates a complementary concentration wave of the labeled complex.

[0115] Assume for illustrative purposes that the electroosmotic (EO)mobility of the buffer is 0.4 cm²/kV-s and the protein has anelectrophoretic (EP) mobility of −0.2 cm²/kV-s. If the EP mobility shiftdue to binding is only 1%, then the EP mobility of the complex is −0.202cm²/kV-s. In a nominal electric field of 250 V/cm along themicrochannel, the velocities of the protein and complex is 0.5 and 0.495mm/s, respectively. For a nominal channel distance of 20 mm between thepoint of reaction and detector location, the time for the protein andcomplex to arrive the detector is 40 and 40.4 s, respectively. The timedifference is therefore 0.4 s between the 2 labeled species. If thistime difference is a significant fraction of the wavelength to achievenoticeable constructive interference, say ¼ (or 90° in phase shift),then a ligand concentration wave of frequency 0.625 Hz (=1/(4×0.4 s)) isneeded. This frequency is practical compared to the response time of atypical electrical controller and a data acquisition rate of 20 Hz.Furthermore, for 0.5 mm/s velocity, this frequency is equivalent toligand injection plugs of 800 μm per cycle spatially. This dimension isalso reasonable when compared to a nominal detector window of ˜50 μm,and a Brownian diffusion length of ˜70 μm under the given flowconditions and the assumption of a protein diffusion constant of 6×10⁻⁷cm²/s.

[0116] Constant Flux Microchip Injector in Quantitative Analysis

[0117] Essentially any analysis in which a starting compound isconverted to a product with a different mobility can be analyzed in amicrofluidic device of the invention. As noted above, essentially anyvelocitogenic assay can be analyzed. One exemplar class of velocitogenicassays includes enzymatic reactions. Kinases are a specific example ofenzymes of this type. Kinases recognize specific polypeptide sequencesand phosphorylate them. Phosphorylation changes the peptide charge, massand structure, and thus the mobilities of the non-phosphorylated andphosphorylated species are different. As a consequence of this change inmobility, substrate and product move at different rates in an appliedfield.

[0118] Enzyme kinetics (i.e., the determination of kcat, Km, and Ki) maybe performed in a microchip capillary electrophoresis experiment bydetermining the extent of conversion of substrate to product.Traditionally, kinetic analyses in a cuvette experiment are performedunder conditions such that the reaction is not substrate limited and theenzymatic turnover is simply a function of the solution conditions andthe inherent catalytic nature of the enzyme. Velocity is irrelevant inthis format. In the microfluidic system, the reaction is homogeneous inthat it occurs in the flowing stream in the capillary. There istypically no surface immobilization of reagents (as described supra, thespecial case where the velocity of a reagent is zero leads to specialconsiderations). Reagents are typically pumped electrokinetically into areaction channel. The field imposed on the flowing stream results in aseparation of each species according to its mobility. In the case wherethe substrate concentration is high relative to the Km of the enzymereaction, the amount of product produced does not depend on theconcentration of substrate. The reaction rate depends only on thereaction conditions and the inherent enzyme reactivity. The signalgenerated in any unit volume is a function of the amount of enzyme inthe reaction mixture, the reaction time, and the electrokinetic mobilityof each species. Unlike the homogeneous cuvette experiment, theelectrokinetic forces used in the microchip format to move reagentsalong the microchannels bias the species concentrations in a reaction.On a microchip, substrate and enzyme flow together through a capillarynetwork, mix, and the substrate is converted to product as the reactionmixtures flows along the length of the mixing channel. Typically influorescence detection, the substrate and product species are bothlabeled with a fluorescent tag. After mixing, the reagents are pumpedelectrokinetically through a portion of the channel passing in front ofthe detector. Samples of the reaction mixture are analyzedquantitatively as substrate and product moieties are separated by theirdifferent electrokinetic mobilities either in the continuous flow modeas described above or by injection followed by separation in anotherchannel.

[0119] In the injection/separation mode, one way to make injections in amicrochip is in a cross or orthogonal injector. In this design, reagentsflow in a fluid path along the length of the applied field. They mix andreact as they flow along the channel. At some distance down the reactionchannel, a perpendicular cross channel is encountered. Injections can bemade from the reaction channel into the separation channel by modulatingthe voltages applied at the end of the capillary length. The injectionvolume is the volume mostly defined by the intersection of theorthogonal channels. The consequence of this type of injection is thatthe amount of reactants and products is a direct reflection of theconcentrations in the reaction channel at the injection point. Theseconcentrations are a function of the solution composition, the enzymereactivity, the reaction time, and the electrokinetic mobilities of thereactants and products. Therefore, in order to determine the substrateand product concentrations, the relative mobilities of each reactant andproduct are determined. Kinetics constant determination requireselectrokinetic correction using the relative mobilities of substrate andproduct as discussed herein.

[0120] Alternatively, an injector that compensates for the differentmobilities of substrate and product in the microchip reaction mixturecan be used. The gated injector is realized in the microchip designwhere the separation channel is collinear with the reaction channel. Inthis case, the electric field for electrokinetic pumping is appliedalong the axis of the reaction channel. The fluid mixture flows alongthis axis but it is directed off the main reaction channel into a sidechannel most of the time, with periodic injection into the collinearseparation channel passing in front of the detector. Buffer orbackground electrolyte from another side channel flows through theseparation channel between the injected aliquots from the reactionchannel. The injections of reaction mixture into the separation channelare pulsed by voltage or current control. The bias imposed by theelectric field pulsing aliquots of reaction mixture to the detectorinfluences the rate at which reagents enter the separation channel. Theresult is that species of highest apparent mobility move fastest intothe separation channel while the low mobility species travel slowly intothe separation channel. This electrokinetic bias in the injection causesspecies that are concentrated in the reaction channel because they moverelatively slowly to map out a smaller volume of injection into theseparation channel.

[0121] Conversely, faster species that are diluted in the reactionchannel map out a proportionately larger volume into the separationchannel due to the higher velocity. Because the same electrokineticforces that result in the concentrating and diluting of analyteconcentrations in the reaction channel also cause the bias in theinjection volumes for the gated injector, the collinear chip injectorcan be used to compensate for the effects of changes in mobility on thedetermination of the extent of reaction in microchips.

[0122] In a simple example in which a substrate with concentration Csand velocity Us is partially converted by an enzyme to a product withconcentration Cp and velocity U_(p), conservation of flux dictates thatJ=C_(p)U_(p)=(yC_(s))U_(s), where y is the fraction of substrateconversion. When this reaction mixture is injected through a gatedinjector into a separation channel, the length of the sample bands forthe unconverted substrate (L_(s)) and for the product (L_(p)) areproportional to their respective velocities, U_(s) and U_(p).L_(s)∝U_(s); L_(p)∝U_(p). The total amount for each species in theinjection volume is the concentration times the volume injected. If A isthe cross-sectional area of the separation channel, then the totalamount of unconverted substrate injected is (y C_(s))L_(s)A, which isproportional to (yC_(s)U_(s)). The total amount of product injected is(C_(p)L_(p)A), which is proportional to (C_(p)U_(p)). Consequently, thetotal amount injected for each species is representative of the flux ofthe species in the reacting channel. Thus, the result of using a gatedinjection is that the extent of chemical conversion can be determinedaccurately without further electrokinetic correction if the total amountof each species can be measured. A “total amount” detector can beaccomplished by setting the detector window (such as a photomultipliertube or PMT slit) spatially wider than the longest sample band length,resulting in peaks whose amplitude is proportional to the amount (aswell as the concentration) one would measure in a non-flowing cuvetteexperiment. Other examples of detectors that report the total amount ofreagent are ones based on total photobleaching and total charge uponcomplete electrochemical conversion. On the other hand, for“concentration” detectors such as a narrow PMT slit compared to thesample band lengths, the extent of reaction still requires the relativemobility correction as disclosed herein because the gated injector doesnot alter the species concentrations in the aliquot.

[0123] Accordingly, in one aspect, the invention provides methods fordispensing representative mixtures by gated injection. In the methods, afirst fluidic mixture is introduced into a first microfluidic channel.The mixture has at least first and second materials; e.g., assaycomponents, reactants or the like, and optionally comprises any numberof additional reaction components. The first and second materials aretransported through the first channel at different velocities, i.e., dueto differences in charge/mass ratios, differing electrophoretic mobilityor the like. An aliquot of the first and second materials is gated(i.e., injected for a selected period of time) into the second channel.The injection can be performed electrokinetically, i.e., by applying avoltage or current difference at the intersection between the first andsecond channel. The precise arrangement of the first and second channelis not critical. For example, the first and second channels optionallycommunicate at a crossing intersection or a T intersection. The relativeamount of first and second materials in the aliquot are proportional tothe flux of first and second materials in the first mixture, therebydispensing a representative mixture of the first and second components.

[0124] Flux is ordinarily conserved in these methods. The flux of thefirst and second components can be the same or different duringelectrokinetic movement. The first or second material can be labeled,and a product resulting from combining the first or second material isoptionally produced. This product is optionally labeled; innon-fluorogenic labeled, the method comprising measuring signal from thealiquot of first or second labeled material, wherein the amount oflabeled material is determined by measuring the signal.

[0125] Modifying Detection Window Size to Analyze VelocitogenicReactions

[0126] As set forth above, the size of the detection region compared tothe size of a sample plug has an effect on the data which is acquired.For a gated injection of a reaction produced on the fly an “amount”detector such as a wide PMT slit (wider than the longest sample plug)results in peaks whose amplitude is proportional to the concentration(or amount) one would measure in a cuvette experiment. For concentrationdetectors (e.g., narrow PMT detection) the concentration is corrected bythe velocity to correctly calculate the percentage of reactantconverted.

[0127] As noted above, a gated injection produces a sample plug in achannel. As the sample plug travels in the channel, the moleculesseparate in the sample plug based upon their respective electrokineticmobilities. As the sample plug passes a detector, all or only a portionof the plug can be detected. If the entire plug is detected, then thetotal amount of any detected species in the plug can be detected. Ifonly a portion of the plug is detected, then the concentration ofmolecules in the detected portion can be determined, by taking velocityinto account as noted herein. If the entire sample plug is detected, avelocity correction does not have to be applied to correctly determinethe amount of product in the plug. Thus, by using gated injection asnoted above, in conjunction with a detection window as wide or widerthan a sample plug passing the detector, amounts of products, reactantsand the like can be determined. Several methods can be used to vary thedetection window size, including varying the slit width where thedetector is a photomultiplier or other similar physical adjustments tothe detector, or by data sampling frequently in time and adding all ofthe data for an entire sample plug.

[0128] Signal Processing, Digital Deconvolution, and Assay ComponentInactivation

[0129] Complex time dependent label signals are observed for reactionsin flowing microfluidic systems. Some of this complexity is due tostacking of charged molecules in the low conductivity running bufferused to separate high conductivity sample plugs and to driveelectroosmotic flow. These complex signals can hinder directinterpretation of data for continuous flow enzyme inhibition or receptorbinding pipettor chip experiments that rely on the use of the high/lowconductivity format for electrokinetic injection.

[0130] Digital signal processing techniques provide a way of simplifyingthe interpretation of data in these types of experiments. Examples ofdata analysis routines that are implemented to simplify datainterpretation include baseline subtraction and masking.

[0131] In baseline subtraction, a series of blanks are injected in acontrol experiment to measure the time dependent baseline, which is thensubtracted from an actual experiment to obtain a difference signal thatis proportional to the degree of inhibition of enzyme activity orreceptor binding.

[0132] In the masking approach, a series of label (e.g., fluorescentdye) injections are made in a control experiment to characterize thetiming of sample plugs as they pass a detection point. For example, thedyes can be injected (e.g., electrokinetically or by pressure injection)into a channel of a microfluidic apparatus and flowed in the channelthrough or past the detection point. The resulting label intensityversus time data is then normalized and subjected to a round offfunction to yield a mask file which has values of 1 corresponding topoints in time at which sample plugs are positioned in front of thedetector and values of 0 for all other times. Multiplication of the maskfile with the data from an actual screening experiment then identifiesthe time windows of interest.

[0133] In both of these approaches, the synchronization of dataacquisition and sample injection is optimally the same for controlexperiments and screening experiments and light source intensities,optics (or other appropriate detector) alignment and injection cycle areoptimally stable over the time course of the experiments. In a preferredembodiment, the labels are fluorescent, although the same approach isused with any label described herein, in conjunction with an appropriatedetector.

[0134] In addition to digital deconvolution techniques, assays areoptionally performed in a format which obviates some of the difficultiesobserved for interpreting assays e.g., utilizing fluidic regionscomprising high conductivity and low conductivity buffers (bracketingcomponents in high or low salt buffers tends to keep components togetherduring electroosmotic flow; see, U.S. Ser. No. 08/761,575 entitled “HighThroughput Screening Assay Systems in Microscale Fluidic Devices” byParce et al. (see also U.S. Ser. No. 08/881,696)). In particular, assaycomponents are optionally deactivated in regions of fluid flowing past adetector. For example, the interpretation of data for continuous flowenzyme inhibition or receptor binding studies are optionally simplifiedby using a running buffer having a pH sufficiently high (or low) todeactivate an assay component in the running buffer, so that signal isonly generated in a sample plug (a region or fluid comprising a highconcentration of sample, typically bracketed by regions of high or lowsalt buffer). Thus, buffers with pH in the range of about 1-5 or about8-14 are useful for inactivating components; for ease of handling,buffers are typically in the range of about pH 3-11.

[0135] Alternatively, other inhibitors of the particular assay componentare optionally added to running buffer, e.g., to inhibit enzyme activityor block receptor binding outside of the sample plug. For example, ionchelators such as EDTA or EGTA are commonly added to reactions toinhibit enzymatic reactions (e.g., where the enzyme requires a Mg⁺⁺ orCa⁺⁺ ion). Similarly, aliquots of high or low temperature buffers, canbe added to inhibit reactions comprising temperature sensitivecomponents. Similarly, heat, cold or light can be applied to the flowingreaction, e.g., by contacting the microfluidic element comprising themicrochannel in which the reaction is run with heat, cold or light. Inthis regard, reactants can be inactivated simply by running thereactants through a region of high electrical resistance (e.g., anarrowed portion of a microfluidic channel). Buffer traversing thisregion of high electrical resistance heats up (a phenomenon referred toas “joule heating”). Accordingly, by selecting current and channelwidth, it is possible to inactivate selected portions of flowingreaction components by joule heating. Thermocycling in microscaledevices utilizing joule heating is described in co-pending applicationU.S. Ser. No. 60/056058, attorney docket number 017646-003800 entitled“ELECTRICAL CURRENT FOR CONTROLLING FLUID TEMPERATURES IN MICROCHANNELS”filed Sep. 2, 1997 by Calvin Chow, Anne R. Kopf-Sill and J. WallaceParce and in 08/977,528, filed Nov. 25, 1997. see also, 08/835,101 andCIP application 09/054,962 by Knapp et al.

[0136] The reaction can proceed for either a selected time in thechannel prior to addition of the inhibitor, or for a selected distancedown the channel. The inhibitor can be added to the entire reactionmixture, or any portion thereof; where the inhibitor is in flowableform, the inhibitor can be added by time or volume gating of theflowable inhibitor.

[0137] In addition to inactivating components in selected regions offlow, inhibitors of reaction such as temperature, pH, ion chelator orthe like are optionally used to deactivate or stop a reaction, e.g.,where the reaction is only to be run for a set period of time.

[0138] Microfluidic Detection Apparatus

[0139] The microfluidic apparatus of the invention often, though notnecessarily, comprise a substrate in which reactants are mixed andanalyzed. A wide variety of suitable substrates for use in the devicesof the invention are described in U.S. Ser. No. 08/761,575, entitled“High Throughput Screening Assay Systems in Microscale Fluidic Devices”by Parce et al. A microfluidic substrate holder is typicallyincorporated into the devices of the invention for holding and/or movingthe substrate during an assay. The substrate holder typically includes asubstrate viewing region for analysis of reactions carried out on thesubstrate. An analyte detector mounted proximal to the substrate viewingregion to detect formation of products and/or passage of reactants alonga portion of the substrate is provided. A computer, operably linked tothe analyte detector, monitors reaction rates by taking velocities andconcentrations of reactants and products into account. An electrokineticcomponent typically provides for movement of the fluids on thesubstrate. Microfluidic devices and systems are also described inAttorney Docket Number 17646-000100, filed Aug. 2, 1996, U.S. Ser. No.08/691,632.

[0140] One of skill will immediately recognize that any, or all, ofthese components are optionally manufactured in separable modular units,and assembled to form an apparatus of the invention. See also, U.S. Ser.No. 08/691,632, supra. In particular, a wide variety of substrateshaving different channels, wells and the like are typically manufacturedto fit interchangeably into the substrate holder, so that a singleapparatus can accommodate, or include, many different substrates adaptedto control a particular reaction. Similarly, computers, analytedetectors and substrate holders are optionally manufactured in a singleunit, or in separate modules which are assembled to form an apparatusfor manipulating and monitoring a substrate. In particular, a computerdoes not have to be physically associated with the rest of the apparatusto be “operably linked” to the apparatus. A computer is operably linkedwhen data is delivered from other components of the apparatus to thecomputer. One of skill will recognize that operable linkage can easilybe achieved using either electrically conductive cable coupled directlyto the computer (e.g., a parallel, serial or modem cables), or usingdata recorders which store data to computer readable media (typicallymagnetic or optical storage media such as computer disks and diskettes,CDs, magnetic tapes, but also optionally including physical media suchas punch cards, vinyl media or the like).

[0141] Substrates and Electrokinetic Modulators

[0142] Suitable substrate materials are generally selected based upontheir compatibility with the conditions present in the particularoperation to be performed by the device. Such conditions can includeextremes of pH, temperature, salt concentration, and application ofelectrical fields. Additionally, substrate materials are also selectedfor their inertness to critical components of an analysis or synthesisto be carried out by the device.

[0143] Examples of useful substrate materials include, e.g., glass,quartz and silicon as well as polymeric substrates, e.g. plastics,particularly polyacrylates. In the case of conductive or semi-conductivesubstrates, it is occasionally desirable to include an insulating layeron the substrate. This is particularly important where the deviceincorporates electrical elements, e.g., electrical fluid directionsystems, sensors and the like. In the case of polymeric substrates, thesubstrate materials may be rigid, semi-rigid, or non-rigid, opaque,semi-opaque or transparent, depending upon the use for which they areintended. For example, devices which include an optical, spectrographic,photographic or visual detection element, will generally be fabricated,at least in part, from transparent materials to allow, or at least,facilitate that detection. Alternatively, transparent windows of, e.g.,glass or quartz, are optionally incorporated into the device for thesetypes of detection elements. Additionally, the polymeric materialsoptionally have linear or branched backbones, and may be crosslinked ornon-crosslinked. Examples of particularly preferred polymeric materialsinclude, e.g., polydimethylsiloxanes (PDMS), polyurethane,polyvinylchloride (PVC) polystyrene, polysulfone, polycarbonate and thelike.

[0144] In certain embodiments, the substrate includes microchannels forflowing reactants and products. At least one of these channels typicallyhas a very small cross sectional dimension, e.g., in the range of fromabout 0.1 μm to about 500 μm. Preferably the cross-sectional dimensionsof the channels is in the range of from about 0.1 to about 200 μm andmore preferably in the range of from about 0.1 to about 100 μm. Inparticularly preferred aspects, each of the channels has at least onecross-sectional dimension in the range of from about 0.1 μm to about 100μm. Although generally shown as straight channels for convenience ofillustration, it will be appreciated that in order to maximize the useof space on a substrate, serpentine, saw tooth or other channelgeometries, are used to incorporate longer channels on less substratearea. Substrates are of essentially any size, with area typicaldimensions of about 1 cm² to 10 cm².

[0145] Manufacturing of these microscale elements into the surface ofthe substrates is generally be carried out by any number ofmicrofabrication techniques that are well known in the art. For example,lithographic techniques are employed in fabricating, e.g., glass, quartzor silicon substrates, using methods well known in the semiconductormanufacturing industries such as photolithographic etching, plasmaetching or wet chemical etching. See, Sorab K. Ghandi, VLSI Principles:Silicon and Gallium Arsenide, NY, Wiley (see, esp. Chapter 10).Alternatively, micromachining methods such as laser drilling, airabrasion, micromilling and the like may be employed. Similarly, forpolymeric substrates, well known manufacturing techniques are used.These techniques include injection molding or stamp molding methodswhere large numbers of substrates may be produced using, e.g., rollingstamps to produce large sheets of microscale substrates or polymermicrocasting techniques where the substrate is polymerized within amicromachined mold. Polymeric substrates are further described inProvisional Patent Application Serial No. 60/015,498, filed Apr. 16,1996 (Attorney Docket No. 017646-002600), and Attorney Docket Number17646-002610, filed Apr. 14, 1997.

[0146] In addition to micromachining methods, printing methods are alsoused to fabricate chambers channels and other microfluidic elements on asolid substrate. Such methods are taught in detail in U.S. Ser. No.08/987,803 by Colin Kennedy, Attorney Docket Number 017646-004400, filedDec. 10, 1997 entitled “Fabrication of Microfluidic Circuits by PrintingTechniques.” In brief, printing methods such as ink-jet printing, laserprinting or other printing methods are used to print the outlines of amicrofluidic element on a substrate, and a cover layer is fixed over theprinted outline to provide a closed microfluidic element.

[0147] The substrates will typically include an additional planarelement which overlays the channeled portion of the substrate enclosingand fluidly sealing the various channels. Attaching the planar coverelement may be achieved by a variety of means, including, e.g., thermalbonding, adhesives or, in the case of certain substrates, e.g., glass,or semi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components. A preferred embodiment is heat lamination,which results in permanent bonding of, e.g., glass substrates. In fact,during heat lamination, the pieces fuse to form a single piece; there isno joint between the pieces, even when viewed by electron microscopy.The planar cover element can additionally be provided with access portsand/or reservoirs for introducing the various fluid elements needed fora particular screen, and for introducing electrodes for electrokineticmovement.

[0148] The introduction of large numbers of individual, discrete volumesof test compounds into the substrate is carried out by any of a numberof methods. For example, micropipettors are used to introduce the testcompounds to the substrate. In one embodiment, an automated pipettor isused. For example, a Zymate XP (Zymark Corporation; Hopkinton, Mass.)automated robot using Microlab 2200 (Hamilton; Reno, Nev.) pipetingstation can be used to transfer parallel samples to regularly spacedwells in a manner similar to transfer of samples to microtiter plates.

[0149] In preferred aspects, an electropipettor is used. An example ofsuch an electropipettor is described in, e.g., U.S. patent applicationSer. No. 08/671,986, filed Jun. 28, 1996 (Attorney Docket No.017646-000500). Generally, this electropipettor utilizes electrokineticor “electroosmotic” fluid direction as described herein, to alternatelysample a number of test compounds, or “subject materials,” and spacercompounds. The pipettor then delivers individual, physically isolatedsample or test compound volumes in subject material regions, in series,into the sample channel for subsequent manipulation within the device.Individual samples are typically separated by a spacer region of lowionic strength spacer fluid. These low ionic strength spacer regionshave higher voltage drop over their length than do the higher ionicstrength subject material or test compound regions, thereby driving theelectrokinetic pumping. On either side of the test compound or subjectmaterial region, which is typically in higher ionic strength solution,are fluid regions referred to as first spacer regions (also referred toas high salt regions on “guard bands”), that contact the interface ofthe subject material regions. These first spacer regions typicallycomprise a high ionic strength solution to prevent migration of thesample elements into the lower ionic strength fluid regions, or secondspacer region, which would result in electrophoretic bias. The use ofsuch first and second spacer regions is described in greater detail inU.S. patent application Ser. No. 08/671,986, filed Jun. 28, 1996,(Attorney Docket No. 017646-000500).

[0150] Alternatively, the channels are individually fluidly connected toa plurality of separate reservoirs via separate channels. The separatereservoirs each contain a separate analyte, reagent, reaction componentor the like, with additional reservoirs being provided, e.g., forappropriate spacer compounds. The test compounds and/or spacer compoundsare transported from the various reservoirs into the sample channelsusing appropriate fluid direction schemes. In either case, it generallyis desirable to separate the discrete sample volumes, or test compounds,with appropriate spacer regions.

[0151] In operation, a fluid first component of a biological system,e.g., a receptor or enzyme, is placed in a first reservoir on thesubstrate. This first component is flowed through a channel past adetection window and toward a waste reservoir. A second component of thebiochemical system, e.g., a ligand or substrate, is concurrently flowedinto the channel, whereupon the first and second components mix and areable to interact. Deposition of these elements within the device arecarried out in a number of ways. For example, the enzyme and substrate,or receptor and ligand solutions introduced into the device through openor sealable access ports in the cover. Alternatively, these componentsare added to their respective reservoirs during manufacture of thedevice. In the case of such pre-added components, it is desirable toprovide these components in a stabilized form to allow for prolongedshelf-life of the device. For example, the enzyme/substrate orreceptor/ligand components are provided within the device in lyophilizedform. Prior to use, these components are easily reconstituted byintroducing a buffer solution into the reservoirs. Alternatively, thecomponents are lyophilized with appropriate buffering salts, wherebysimple water addition is all that is required for reconstitution.

[0152] Flowing and direction of fluids within the microscale fluidicdevices may be carried out by a variety of methods. For example, thedevices may include integrated microfluidic structures, such asmicropumps and microvalves, or external elements, e.g., pumps andswitching valves, for the pumping and direction of the various fluidsthrough the device. Examples of microfluidic structures are describedin, e.g., U.S. Pat. Nos. 5,271,724, 5,277,556, 5,171,132, and 5,375,979.See also, Published U.K. Patent Application No. 2 248 891 and PublishedEuropean Patent Application No. 568 902.

[0153] Although microfabricated fluid pumping and valving systems may bereadily employed in the devices of the invention, the cost andcomplexity associated with their manufacture and operation can generallyprohibit their use in mass-produced disposable devices as are envisionedby the present invention. Furthermore, the velocity of components insuch systems is driven by overall fluid flow, making consideration ofvelocity less relevant in these systems (there is no electrophoreticcomponent of velocity in a pure pressure-driven system). For thatreason, the devices of the invention will typically include anelectroosmotic fluid direction system. Such fluid direction systemscombine the elegance of a fluid direction system devoid of moving parts,with an ease of manufacturing, fluid control and disposability. Examplesof particularly preferred electroosmotic fluid direction systemsinclude, e.g., those described in International Patent Application No.WO 96/04547 to Ramsey et al., as well as U.S. Ser. No. 08/761,575 byParce et al.

[0154] In brief, these fluidic control systems typically includeelectrodes disposed within reservoirs that are placed in fluidconnection with the channels fabricated into the surface of thesubstrate. The materials stored in the reservoirs are transportedthrough the channel system delivering appropriate volumes of the variousmaterials to one or more regions on the substrate in order to carry outa desired screening assay.

[0155] Fluid transport and direction is accomplished throughelectroosmosis or electrokinesis. In brief, when an appropriate fluid isplaced in a channel or other fluid conduit having functional groupspresent at the surface, those groups can ionize. For example, where thesurface of the channel includes hydroxyl functional groups at thesurface, protons can leave the surface of the channel and enter thefluid. Under such conditions, the surface will possess a net negativecharge, whereas the fluid will possess an excess of protons or positivecharge, particularly localized near the interface between the channelsurface and the fluid. By applying an electric field along the length ofthe channel, cations will flow toward the negative electrode. Movementof the positively charged species in the fluid pulls the solvent withthem.

[0156] To provide appropriate electric fields, the system generallyincludes a voltage controller that is capable of applying selectablevoltage levels, simultaneously, to each of the reservoirs, includingground. Such a voltage controller can be implemented using multiplevoltage dividers and multiple relays to obtain the selectable voltagelevels. Alternatively, multiple, independent voltage sources may beused. The voltage controller is electrically connected to each of thereservoirs via an electrode positioned or fabricated within each of theplurality of reservoirs. In one embodiment, multiple electrodes arepositioned to provide for switching of the electric field direction in amicrochannel, thereby causing the analytes to travel a longer distancethan the physical length of the microchannel.

[0157] Substrate materials are also selected to produce channels havinga desired surface charge. In the case of glass substrates, the etchedchannels will possess a net negative charge resulting from the ionizedhydroxyls naturally present at the surface. Alternatively, surfacemodifications may be employed to provide an appropriate surface charge,e.g., coatings, derivatization, e.g., silanation, or impregnation of thesurface to provide appropriately charged groups on the surface. Examplesof such treatments are described in, e.g., Provisional PatentApplication Serial No. 60/015,498, filed Apr. 16, 1996 (Attorney DocketNo. 017646-002600). See also, Attorney Docket Number 17646-002610, filedApr. 14, 1997.

[0158] Modulating voltages are then concomitantly applied to the variousreservoirs to affect a desired fluid flow characteristic, e.g.,continuous or discontinuous (e.g., a regularly pulsed field causing theflow to oscillate direction of travel) flow of receptor/enzyme,ligand/substrate toward the waste reservoir with the periodicintroduction of test compounds. Particularly, modulation of the voltagesapplied at the various reservoirs can move and direct fluid flow throughthe interconnected channel structure of the device in a controlledmanner to effect the fluid flow for the desired screening assay andapparatus.

[0159] Detectors and Labels

[0160] A “label” is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention includefluorescent dyes (e.g., fluorescein isothiocyanate, texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P,³³P, etc.), enzymes (e.g., horse-radish peroxidase, alkaline phosphataseetc.) colorimetric labels such as colloidal gold or colored glass orplastic (e.g. polystyrene, polypropylene, latex, etc.) beads. The labelmay be coupled directly or indirectly to the a component of the assayaccording to methods well known in the art. As indicated above, a widevariety of labels may be used, with the choice of label depending onsensitivity required, ease of conjugation with the compound, stabilityrequirements, available instrumentation, and disposal provisions.Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, or cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody (see,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY;and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY for a general discussion of how to make and useantibodies). The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol.

[0161] In some embodiments, a first and second label on the same ordifferent components interact when in proximity (e.g., due tofluorescence resonance transfer), and the relative proximity of thefirst and second labels is determined by measuring a change in theintrinsic fluorescence of the first or second label. For example, theemission of a first label is sometimes quenched by proximity of thesecond label. Many appropriate interactive labels are known. Forexample, fluorescent labels, dyes, enzymatic labels, and antibody labelsare all appropriate. Examples of interactive fluorescent label pairsinclude terbium chelate and TRITC (tetrarhodamine isothiocyanate),europium cryptate and Allophycocyanin, DABCYL and EDANS and many othersknown to one of skill. Similarly, two colorimetric labels can result incombinations which yield a third color, e.g., a blue emission inproximity to a yellow emission provides an observed green emission. Withregard to preferred fluorescent pairs, there are a number offluorophores which are known to quench one another. Fluorescencequenching is a bimolecular process that reduces the fluorescence quantumyield, typically without changing the fluorescence emission spectrum.Quenching can result from transient excited state interactions,(collisional quenching) or, e.g., from the formation of nonfluorescentground state species. Self quenching is the quenching of one fluorophoreby another; it tends to occur when high concentrations, labelingdensities, or proximity of labels occurs. Fluorescent resonance energytransfer (FRET) is a distance dependent excited state interaction inwhich emission of one fluorophore is coupled to the excitation ofanother which is in proximity (close enough for an observable change inemissions to occur). Some excited fluorophores interact to formexcimers, which are excited state dimers that exhibit altered emissionspectra (e.g., phospholipid analogs with pyrene sn-2 acyl chains); see,Haugland (1996) Handbook of Fluorescent Probes and Research ChemicalsPublished by Molecular Probes, Inc., Eugene, Oreg. e.g., at chapter 13).

[0162] Detectors for detecting labeled compounds are known to those ofskill in the art. Thus, for example, where the label is a radioactivelabel, means for detection include a scintillation counter orphotographic film as in autoradiography. Where the label is afluorescent label, it may be detected by exciting the fluorochrome withthe appropriate wavelength of light and detecting the resultingfluorescence. The fluorescence may be detected visually, by means ofphotographic film, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers, phototubes, photodiodes orthe like. Similarly, enzymatic labels are detected by providing theappropriate substrates for the enzyme and detecting the resultingreaction product. Finally simple calorimetric labels may be detectedsimply by observing the color associated with the label. This is doneusing a spectrographic device, e.g., having an appropriate grating,filter or the like allowing passage of a particular wavelength of light,and a photodiode, or other detector for converting light to anelectronic signal, or for enhancing visual detection.

[0163] The substrate includes a detection window or zone at which asignal is monitored. For example, reactants or assay components arecontacted in a microfluidic channel in a first region, and subsequentlyflowed into a second channel region comprising a detection window orregion. The first and second channel region are optionally part of asingle channel, but can also be separate channels, e.g., which are influid connection. This detection window or region typically includes alight or radiation transparent cover allowing visual or opticalobservation and detection of the assay results, e.g., observation of acolorometric, fluorometric or radioactive response, or a change in thevelocity of colorometric, fluorometric or radioactive component.Detectors detect a labeled compound. Example detectors includespectrophotometers, photodiodes, microscopes, scintillation counters,cameras, film and the like, as well as combinations thereof. Examples ofsuitable detectors are widely available from a variety of commercialsources known to persons of skill.

[0164] In one aspect, monitoring of the signals at the detection windowis achieved using an optical detection system. For example, fluorescencebased signals are typically monitored using, e.g., in laser activatedfluorescence detection systems which employ a laser light source at anappropriate wavelength for activating the fluorescent indicator withinthe system. Fluorescence is then detected using an appropriate detectorelement, e.g., a photomultiplier tube (PMT). Similarly, for screensemploying colorometric signals, spectrophotometric detection systems maybe employed which detect a light source at the sample and provide ameasurement of absorbance or transmissivity of the sample. See also, ThePhotonics Design and Applications Handbook, books 1, 2, 3 and 4,published annually by Laurin Publishing Co., Berkshire Common, P.O. Box1146, Pittsfield, Mass. for common sources for optical components.

[0165] In alternative aspects, the detection system comprisesnon-optical detectors or sensors for detecting a particularcharacteristic of the system disposed within detection window 116. Suchsensors may include temperature (useful, e.g., when a reaction producesor absorbs heat), conductivity, potentiometric (pH, ions), amperometric(for compounds that may be oxidized or reduced, e.g., O₂, H₂O₂, I₂,oxidizable/reducible organic compounds, and the like).

[0166] Alternatively, schemes similar to those employed for theenzymatic system may be employed, where there is a signal that reflectsthe interaction of the receptor with its ligand. For example, pHindicators which indicate pH effects of receptor-ligand binding may beincorporated into the device along with the biochemical system, i.e., inthe form of encapsulated cells, whereby slight pH changes resulting frombinding can be detected. See Weaver, et al., Bio/Technology (1988)6:1084-1089. Additionally, one can monitor activation of enzymesresulting from receptor ligand binding, e.g., activation of kinases, ordetect conformational changes in such enzymes upon activation, e.g.,through incorporation of a fluorophore which is activated or quenched bythe conformational change to the enzyme upon activation.

[0167] One conventional system carries light from a specimen field to acooled charge-coupled device (CCD) camera. A CCD camera includes anarray of picture elements (pixels). The light from the specimen isimaged on the CCD. Particular pixels corresponding to regions of thesubstrate are sampled to obtain light intensity readings for eachposition. Multiple positions are processed in parallel and the timerequired for inquiring as to the intensity of light from each positionis reduced. Many other suitable detection systems are known to one ofskill.

[0168] Assays

[0169] In the assays of the invention, a first reactant or assaycomponent is contacted to a second reactant or product, typically toform a product. The reactants or components can be elements ofessentially any assay which is adaptable to a flowing format; thus,while often described in terms of enzyme-substrate or receptor-ligandinteractions, it will be understood that the reactants or componentsherein can comprise a moiety derived from any of a wide variety ofcomponents, including, antibodies, antigens, ligands, receptors,enzymes, enzyme substrates, amino acids, peptides, proteins,nucleosides, nucleotides, nucleic acids, fluorophores, chromophores,biotin, avidin, organic molecules, monomers, polymers, drugs,polysaccharides, lipids, liposomes, micelles, toxins, biopolymers,therapeutically active compounds, molecules from biological sources,blood constituents, cells or the like. No attempt is made herein todescribe how known assays utilizing these components are practiced. Awide variety of microfluidic assays are practiced using thesecomponents. See, e.g., U.S. Ser. No. 08/761,575 entitled “HighThroughput Screening Assay Systems in Microscale Fluidic Devices” byParce et al. (see also U.S. Ser. No. 08/881,696).

[0170] As used herein, the phrase “biochemical system” generally refersto a chemical interaction that involves molecules of the type generallyfound within living organisms. Such interactions include the full rangeof catabolic and anabolic reactions which occur in living systemsincluding enzymatic, binding, signalling and other reactions. Further,biochemical systems, as defined herein, also include model systems whichare mimetic of a particular biochemical interaction. Examples ofbiochemical systems of particular interest in practicing the presentinvention include, e.g., receptor-ligand interactions, enzyme-substrateinteractions, cellular signaling pathways, transport reactions involvingmodel barrier systems (e.g., cells or membrane fractions) forbioavailability screening, and a variety of other general systems.Cellular or organismal viability or activity may also be screened usingthe methods and apparatuses of the present invention, e.g., intoxicology studies. Biological materials which are assayed include, butare not limited to, cells, cellular fractions (membranes, cytosolpreparations, etc.), agonists and antagonists of cell membrane receptors(e.g., cell receptor-ligand interactions such as e.g., transferrin,c-kit, viral receptor ligands (e.g., CD4-HIV), cytokine receptors,chemokine receptors, interleukin receptors, immunoglobulin receptors andantibodies, the cadherein family, the integrin family, the selectinfamily, and the like; see, e.g., Pigott and Power (1993) The AdhesionMolecule FactsBook Academic Press New York and Hulme (ed) ReceptorLigand Interactions A Practical Approach Rickwood and Hames (serieseditors) IRL Press at Oxford Press NY), toxins and venoms, viralepitopes, hormones (e.g., opiates, steroids, etc.), intracellularreceptors (e.g. which mediate the effects of various small ligands,including steroids, thyroid hormone, retinoids and vitamin D; forreviews see, e.g., Evans (1988) Science, 240:889-895; Ham and Parker(1989) Curr. Opin. Cell Biol, 1:503-511; Burnstein et al. (1989), Ann.Rev. Physiol., 51:683-699; Truss and Beato (1993) Endocr. Rev.,14:459-479), peptides, retro-inverso peptides, polymers of α-, β-, or ω-amino acids (D- or L-), enzymes, enzyme substrates, cofactors, drugs,lectins, sugars, nucleic acids (both linear and cyclic polymerconfigurations), oligosaccharides, proteins, phospholipids andantibodies. Synthetic polymers such as heteropolymers in which a knowndrug is covalently bound to any of the above, such as polyurethanes,polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,polyarylene sulfides, polysiloxanes, polyimides, and polyacetates arealso assayed. Other polymers are also assayed using the systemsdescribed herein, as would be apparent to one of skill upon review ofthis disclosure. One of skill will be generally familiar with thebiological literature. For a general introduction to biological systems,see, Berger and Kimmel, Guide to Molecular Cloning Techniques, Methodsin Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual(2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring HarborPress, NY, (Sambrook); Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (through 1998Supplement) (Ausubel); Watson et al. (1987) Molecular Biology of theGene, Fourth Edition The Benjamin/Cummings Publishing Co., Menlo Park,Calif.; Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, NY; Alberts et al. (1989) Molecular Biology of the CellSecond Edition Garland Publishing, NY; Pattison (1994) Principles andPractice of Clinical Virology; Darnell et al., (1990) Molecular CellBiology second edition, Scientific American Books, W. H. Freeman andCompany; Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck &Co., Rahway, N.J.; Harrison's Principles of Internal Medicine,Thirteenth Edition, Isselbacher et al. (eds). (1994) Lewin Genes, 5thEd., Oxford University Press (1994); The “Practical Approach” Series ofBooks (Rickwood and Hames (series eds.) by IRL Press at OxfordUniversity Press, NY; The “FactsBook Series” of books from AcademicPress, NY, ; Product information from manufacturers of biologicalreagents and experimental equipment also provide information useful inassaying biological systems. Such manufacturers include, e.g., the SIGMAchemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.),Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research,Inc.^(•, GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill.)

[0171] In order to provide methods and devices for screening compoundsfor effects on biochemical systems, the present invention generallyincorporates model in vitro systems which mimic a given biochemicalsystem in vivo for which effector compounds are desired. The range ofsystems against which compounds can be screened and for which effectorcompounds are desired, is extensive. For example, compounds areoptionally screened for effects in blocking, slowing or otherwiseinhibiting key events associated with biochemical systems whose effectis undesirable. As described supra, the effects of velocity of thecomponents are corrected for to provide accurate determinations of therates of these key events.

[0172] For example, assay compounds are optionally screened for theirability to block systems that are responsible, at least in part, for theonset of disease or for the occurrence of particular symptoms ofdiseases, including, e.g., hereditary diseases, cancer, bacterial orviral infections and the like. Compounds which show promising results inthese screening assay methods can then be subjected to further testingto identify effective pharmacological agents for the treatment ofdisease or symptoms of a disease. Using the data correction methodsdescribed herein, the effects of assay compounds on biochemical systemsis properly determined. For example, the binding properties of a testmolecule to a target, or the effects of an enzyme modulator are easilydetermined using the methods herein.

[0173] Alternatively, compounds can be screened for their ability tostimulate, enhance or otherwise induce biochemical systems whosefunction is believed to be desirable, e.g., to remedy existingdeficiencies in a patient. Furthermore, as described extensively supra,enzyme activity levels (which can be diagnostic of diseases) arecorrectly determined using the methods herein.

[0174] Once a model system is selected, batteries of test compounds canbe applied against these model systems. By identifying those testcompounds that have an effect on the particular biochemical system, invitro, one can identify potential effectors of that system, in vivo.

[0175] In one form, the biochemical system models employed in themethods and apparatuses of the present invention will screen for aneffect of a an assay compound on an interaction between two or morecomponents of a biochemical system, e.g., receptor-ligand interaction,enzyme-substrate interaction, and the like. In this form, thebiochemical system model will typically include the two normallyinteracting components of the system for which an effector is sought,e.g., the receptor and its ligand or the enzyme and its substrate.

[0176] Determining whether a test compound has an effect on thisinteraction then involves contacting the system with an assay compoundand assaying for the functioning of the system, e.g., receptor-ligandbinding or substrate turnover. The assayed function is then compared toa control, e.g., the same reaction in the absence of the test compoundor in the presence of a known effector, taking proper steps to correctfor velocity of components as described supra. Typically, such assaysinvolve the measurement of a parameter of the biochemical system. By“parameter of the biochemical system” is meant some measurable evidenceof the system's functioning, e.g., the presence or absence of a labeledgroup or a change in molecular weight (e.g., in binding reactions,transport screens), the presence or absence of a reaction product orsubstrate (in substrate turnover measurements), or an alteration inelectrophoretic mobility (detected, e.g., by a change in signal from adetector in the system).

[0177] Although described in terms of two-component biochemical systems,the methods and apparatuses may also be used to screen for effectors ofmuch more complex systems, where the result or end product of the systemis known and assayable at some level, e.g., enzymatic pathways, cellsignaling pathways and the like. Alternatively, the methods andapparatuses described herein are optionally used to screen for compoundsthat interact with a single component of a biochemical system, e.g.,compounds that specifically bind to a particular biochemical compound,e.g., a receptor, ligand, enzyme, nucleic acid, structuralmacromolecule, etc. In all of these instances, the ability to correctlymeasure binding reactions, product production rates, assay componentconcentrations and the like, using the methods herein, makes the assaymore predictive and representative.

[0178] Biochemical system models are also embodied in whole cellsystems. For example, where one is seeking to screen test compounds foran effect on a cellular response, whole cells are optionally utilized.Modified cell systems are employed in the systems encompassed herein.For example, chimeric reporter systems are optionally employed asindicators of an effect of a test compound on a particular biochemicalsystem. Chimeric reporter systems typically incorporate a heterogenousreporter system integrated into a signaling pathway which signals thebinding of a receptor to its ligand. For example, a receptor is fused toa heterologous protein, e.g., an enzyme whose activity is readilyassayable. Activation of the receptor by ligand binding then activatesthe heterologous protein, which then allows for detection. Thus, thesurrogate reporter system produces an event or signal which is readilydetectable, thereby providing an assay for receptor/ligand binding.Examples of such chimeric reporter systems have been previouslydescribed in the art. An example is the common chloramphenicol acetyltransferase (CAT) assay.

[0179] Additionally, where one is screening for bioavailability, e.g.,transport, biological barriers are optionally included. The term“biological barriers” generally refers to cellular or membranous layerswithin biological systems, or synthetic models thereof. Examples of suchbiological barriers include the epithelial and endothelial layers, e.g.vascular endothelia and the like.

[0180] Biological responses are often triggered and/or controlled by thebinding of a receptor to its ligand. For example, interaction of growthfactors, e.g., EGF, FGF, PDGF, etc., with their receptors stimulates awide variety of biological responses including, e.g., cell proliferationand differentiation, activation of mediating enzymes, stimulation ofmessenger turnover, alterations in ion fluxes, activation of enzymes,changes in cell shape and the alteration in genetic expression levels.Accordingly, control of the interaction of the receptor and its ligandmay offer control of the biological responses caused by thatinteraction.

[0181] Accordingly, in one aspect, the present invention will be usefulin screening for, or testing the activity of, compounds that affect aninteraction between a receptor molecule and its ligands. As used herein,the term “receptor” generally refers to one member of a pair ofcompounds which specifically recognize and bind to each other. The othermember of the pair is termed a “ligand.” Thus, a receptor/ligand pairmay include a typical protein receptor, usually membrane associated, andits natural ligand, e.g., another protein or small molecule.Receptor/ligand pairs can include antibody/antigen binding pairs,complementary nucleic acids, nucleic acid associating proteins and theirnucleic acid ligands. A large number of specifically associatingbiochemical compounds are well known in the art and can be utilized inpracticing the present invention.

[0182] Traditionally, methods for screening for effectors of areceptor/ligand interaction have involved incubating a receptor/ligandbinding pair in the presence of a test compound. The level of binding ofthe receptor/ligand pair is then compared to negative and/or positivecontrols. Where a decrease in normal binding is seen, the test compoundis determined to be an inhibitor of the receptor/ligand binding. Wherean increase in that binding is seen, the test compound is determined tobe an enhancer or inducer of the interaction. The methods of correctingfor velocity and other effects as noted herein provide for correctdetermination of these parameters.

[0183] Typically, effectors of an enzyme's activity toward its substrateare screened by contacting the enzyme with a substrate in the presenceand absence of the compound to be screened and under conditions optimalfor detecting changes in the enzyme's activity. After a set time forreaction, the mixture is assayed for the presence of reaction productsor a decrease in the amount of substrate. The amount of substrate thathas been catalyzed is them compared to a control, i.e., enzyme contactedwith substrate in the absence of test compound or presence of a knowneffector. As above, a compound that reduces the enzymes activity towardits substrate is termed an “inhibitor,” whereas a compound thataccentuates that activity is termed an “inducer.” Again, using the datacorrection methods herein, a correct determination of whether acomponent is an inhibitor, an inducer, or irrelevant to the system canmore easily be determined.

[0184] The various methods encompassed by the present inventionoptionally involve the serial or parallel introduction of one or aplurality of assay components into a microfluidic device. Once in thedevice, the assay component is screened for effect on a biological orchemical system using a serial or parallel assay format.

[0185] Assay components are optionally screened for their ability toaffect a particular biochemical or chemical system. Assay components caninclude a wide variety of different compounds, including chemicalcompounds, mixtures of chemical compounds, e.g., polysaccharides, smallorganic or inorganic molecules, biological macromolecules, e.g.,peptides, proteins, nucleic acids, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal cells or tissues,naturally occurring or synthetic compositions. Depending upon theparticular embodiment being practiced, the assay components are providedfrom a source of assay components, e.g., injected, free in solution,optionally attached to a carrier, a solid support, e.g., beads or thelike. A number of suitable supports are employed for immobilization ofthe assay components. Examples of suitable solid supports includeagarose, cellulose, dextran (commercially available as, i.e., Sephadex,Sepharose) carboxymethyl cellulose, polystyrene, polyethylene glycol(PEG), filter paper, nitrocellulose, ion exchange resins, plastic films,glass beads, polyaminemethylvinylether maleic acid copolymer, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc.Additionally, for the methods and apparatuses described herein, testcompounds are screened individually, or in groups. Group screening isparticularly useful where hit rates for effective test compounds areexpected to be low such that one would not expect more than one positiveresult for a given group. Alternatively, such group screening is usedwhere the effects of different test compounds are differentiallydetected in a single system, e.g., through electrophoretic separation ofthe effects, or differential labelling which enables separate detection.

[0186] Assay components are commercially available, or derived from anyof a variety of biological sources apparent to one of skill and asdescribed, supra. In one aspect, a tissue homogenate or blood samplefrom a patient is tested in the assay systems of the invention. Forexample, in one aspect, blood is tested for the presence or activity ofa biologically relevant molecule. For example, the presence and activitylevel of an enzyme are detected by supplying and enzyme substrate to thebiological sample and detecting the formation of a product using anassay systems of the invention. Similarly, the presence of infectiouspathogens (viruses, bacteria, fungi, or the like) or cancerous tumorscan be tested by monitoring binding of a labeled ligand to the pathogenor tumor cells, or a component of the pathogen or tumor such as aprotein, cell membrane, cell extract or the like, or alternatively, bymonitoring the presence of an antibody against the pathogen or tumor inthe patient's blood. For example, the binding of an antibody from apatient's blood to a viral protein such as an HIV protein is a commontest for monitoring patient exposure to the virus. Many assays fordetecting pathogen infection are well known, and are adapted to theassay systems of the present invention.

[0187] Biological samples are derived from patients using well knowntechniques such as venipuncture or tissue biopsy. Where the biologicalmaterial is derived from non-human animals, such as commerciallyrelevant livestock, blood and tissue samples are conveniently obtainedfrom livestock processing plants. Similarly, plant material used in theassays of the invention are conveniently derived from agricultural orhorticultural sources. Alternatively, a biological sample can be from acell or blood bank where tissue and/or blood are stored, or from an invitro source such as a culture of cells. Techniques and methods forestablishing a culture of cells for use as a source for biologicalmaterials are well known to those of skill in the art. Freshney Cultureof Animal Cells, a Manual of Basic Technique, Third Edition Wiley-Liss,New York (1994) provides a general introduction to cell culture.

[0188] In addition to biological systems, the apparatus and methods ofthe invention are adaptable to chemical synthetic approaches. Forexample chemical synthetic methods for making proteins, nucleic acids,amino acids, polymers, organic compounds and the like are well known. Ingeneral, most chemical synthetic protocols employ fluid mixing to mixreactants, reagents and the like. As applied to the present invention, asource of reactants, reagents or the like is fluidly coupled to amicrofluidic channel. The reactants or reagents, which optionallycomprise labels, are mixed in a microchannel. After mixing, reactionrates, product concentrations, reactant concentrations or the like areeasily determined using the methods described herein. Representativemixtures can be aliquoted from one channel into a different channel forsubsequent analysis, e.g., using the time-gated methods described supra.No attempt is made to describe all of the possible reactants, reactionsor products which can be employed in the methods and devices of theinvention; it is presumed that one of skill is generally familiar withsuch known methods, and that, upon review of this disclosure, couldadapt these known assays to the present system.

[0189] As described above, the screening methods of the presentinvention are generally carried out in microfluidic devices or“microlaboratory systems,” which allow for integration of the elementsrequired for performing the assay, automation, and minimal environmentaleffects on the assay system, e.g., evaporation, contamination, humanerror, or the like. A number of devices for carrying out the assaymethods of the invention are described in substantial detail herein.However, it will be recognized that the specific configuration of thesedevices will generally vary depending upon the type of assay and/orassay orientation desired. For example, in some embodiments, thescreening methods of the invention can be carried out using amicrofluidic device having two intersecting channels. For more complexassays or assay orientations, multichannel/intersection devices areoptionally employed. The small scale, integratability and self-containednature of these devices allows for virtually any assay orientation to berealized within the context of the microlaboratory system. In addition,it will be realized that the data correction methods herein areapplicable to flowing systems generally, and not simply in microfluidicsystems.

[0190] Computers

[0191] Typically, when using a detection device such as that describedherein, data thus obtained is stored and analyzed using a computer. Thismay be accomplished by digitizing an image from the detection device andstoring the image on a computer-readable medium. This is normallyaccomplished by storing the data representing the digitized image in adatabase, spreadsheet file, or similar storage vehicle on a computer'sstorage media. A computer operably linked to the analyte detector istherefore provided. The computer is coupled to the microfluidic deviceusing cables to connect the computer to the data detection device.Alternatively, the data may be recorded on a data collection device andtransported (e.g., on a computer-readable storage medium) to thecomputer for processing. Software on the computer determines the rate offormation of the analyte, correcting for the effects of the motion ofthe analyte. This is done, for example, by determining or collating thevelocities of one or more components and the concentrations of one ormore components and calculating the rate of formation of one or morecomponents, while correcting for each components' velocity.

[0192] A variety of commercially available hardware and software isavailable for digitizing, storing, and analyzing a signal or image suchas that generated by the microfluidic device described herein.Typically, a computer commonly used to transform signals from thedetection device into reaction rates will be a PC-compatible computer(e.g., having a central processing unit (CPU) compatible with x86 CPUs,and running an operating system such as DOS™, OS/2 Warp™, WINDOWS/NT™,or WINDOWS 95™), a Macintosh™ (running MacOS™), or a UNIX workstation(e.g., a SUN™ workstation running a version of the Solaris™ operatingsystem, or PowerPC™ workstation) are all commercially common, and knownto one of skill in the art. Data analysis software on the computer isthen employed to determine the rate of formation of the analyte inmotion. Software for determining reaction rates is available, or caneasily be constructed by one of skill using a standard programminglanguage such as Visual Basic, Fortran, Basic, Java, or the like. Thesoftware is designed to determine velocities, concentrations, fluxrelationships and the like, as described herein.

[0193] In general, software designed to perform data manipulations willinclude several common steps. FIG. 4B illustrates the steps performed incalculating a concentration profile along a microfluidic channel for acontinuous flow binding assay as a function of time for a givenassociation constant (K_(a)) The process illustrated by FIG. 4B beginsat step 400 with the acquisition of the data from the detection device.Data thus acquired is then stored in a database, spreadsheet file, orsimilar construct (step 405). As noted, these steps may be carried outremotely from the computer system used to analyze the acquired data,with the acquired data being transferred to the computer system usingremovable media, network, or other such mechanism. The structures usedto store the data (e.g., arrays) are then initialized (step 410). Thisincludes calculating row indices and initializing the time index, andzeroing-out the concentration arrays. Test parameters are then read infrom storage on the computer (step 415). This includes ranges for thevariables, including the time increment between measurements. Theseoperations need not be performed in this order, as they merely set upthe variables considered in performing the calculations outlined supra.Next equilibrium concentrations are calculated (step 420). Theconcentration profile information generated by this step is then outputto the database (step 425). The timing signal value corresponding to theconcentration profile information is also output to the database (step430).

[0194] Next, at step 435, flow conditions are used to calculate motionof the various chemical species involved in the test being analyzed.This, in effect, corresponds to the motion of the various chemicalspecies down the microfluidic channel. The changes are reflected in thevariable representing the concentrations of each of the chemicalspecies. At step 436, new equilibrium concentrations are calculated foreach of the chemical species. Again, concentration profile informationand corresponding timing signal information generated by the equilibriumcalculations are output to the database (steps 437 and 438,respectively). As noted, each test is broken up into time increments.Analysis of the test finishes when the number of time increments equalsthe duration of the test (step 440). Otherwise, the index representingthe time elapsed is incremented (also represented by step 440) and steps435-438 repeated, as illustrated in FIG. 4B.

[0195]FIG. 4C illustrates an alternative set of steps according to thepresent invention for calculating a concentration profile along amicrofluidic channel for a continuous flow binding assay as a functionof time for a given association constant (K_(a)). The processillustrated by FIG. 4C begins at step 445 with the acquisition of thedata from the detection device. Data thus acquired is then stored in adatabase, spreadsheet file, or similar construct (step 450). As noted,these steps may be carried out remotely from the computer system used toanalyze the acquired data, with the acquired data being transferred tothe computer system using removable media, network, or other suchmechanism. Test parameters are then read in from storage on the computer(step 455), including ranges for the considered variables, including thetime increment between measurements. The structures used to store thedata (e.g., arrays) are then initialized (step 460), includingcalculating row indices and initializing the time index, and zeroing-outthe concentration arrays. As before, these operations need not beperformed in this order. Next, initial concentration profile informationis output to the database (step 465). The timing signal valuecorresponding to the concentration profile information is also output tothe database (step 470).

[0196] Next, at step 475, motion of the chemical species is calculated,corresponding to the motion of the various chemical species down themicrofluidic channel. These changes are reflected in the variablerepresenting the concentrations of each of the chemical species. At step476, enzyme reactions are calculated. Again, concentration profileinformation and corresponding timing signal information generated by theequilibrium calculations are output to the database (steps 477 and 478,respectively). As noted, each test is broken up into time increments.Analysis of the test finishes when the number of time increments equalsthe duration of the test (step 480). Otherwise, the index representingthe time elapsed is incremented (also represented by step 480) and steps475-478 repeated, as illustrated in FIG. 4C.

[0197] Exemplary spreadsheet macro software is provided in Appendix Aand Appendix B.

EXAMPLES

[0198] The following examples are offered to illustrate, but not tolimit the present invention.

Example 1

[0199] Monitoring Flux in a Microchannel

[0200] In a given microchannel of a microfluidic device, the flux (J),with units of molecules/(cross sectional area×time), is equal to thevelocity of the molecules under consideration (U) times theconcentration of molecules (C); J=U×C. Flux is conserved in themicrochannel under consideration. In other words, the sum of the numberof analyte molecules (enzymes, substrates and products, or ligands andligand partners) times the velocity of the components is constant.

[0201] Enzyme-Substrate Assay

[0202] For example, in the following chemical system, a substrate and anenzyme are mixed at point M, and travel along a microchannel with lengthL to a detection point. The detector at the detection point can observeproduct molecules formed from the substrate, and/or substrate moleculesand/or enzyme molecules as described in FIG. 4A. The enzyme (E) andsubstrate (S) are mixed and react to convert a small portion of thesubstrate into a product (P). In a preferred embodiment, the product isflorescent, and easily detectable, e.g., using a photodiode,photomultiplier, a spectrometer, or the like.

[0203] In the case in which P and S have the same mobility, or in astationary system, a concentration balance for the reacted and unreactedcomponents is described by a simple concentration balance.

[0204] [E]×T_(LS)×k=[S]_(converted)=C_(P), where [E], [S] and C_(P) areenzyme, substrate and product concentrations, respectively, in units ofmolecules per volume; T_(LS) is the transit time of substrate betweenmixing and detection points or the reaction time, which is equivalent tothe length for reaction divided by the velocity of substrate, L/U_(s).The reaction constant, k, has units of molecules of product permolecules of enzyme per time.

[0205] Analyzing with the flux being conserved in a system where theproduct velocity and substrate velocity are not necessarily identicalresults in: Flux(J)=[E]×T_(LS)×k×U_(s)=[S]_(converted)×U_(s)=U_(p)×C_(P), where U_(p) isthe product velocity. Rearranging and writing transit time of substrateas L/U, results in: [E]×L/U_(s)×k×U_(s)=U_(p)×C_(P). Then:[E]/U_(p)×L×k=C_(P). Substituting transit time, T_(LP) for product givesthe non-intuitive result that product concentration is proportional tothe transit time of the product, not substrate as might be extrapolatedfrom the stationary or non-mobility changing case above:[E]×T_(LP)×k=C_(P).

[0206] Joined Reactants Assay

[0207] In a binding assay where the binding of two molecules in areaction system results in a product with a change in mobility, asimilar analysis can be undertaken. For example when streptavidin (SA),a large molecule, binds to biotin, it changes the mobility of thelabeled biotin. In one embodiment, spacer molecules (T10) are placedbetween the B and SA molecules to prevent quenching of B when SA isbound. Thus, both B and product molecules (B-SA) are fluorescent. Thesimple device depicted in FIG. 5 can be used for mixing and detection ofthe substrates and products, optionally further including a detector,computer, or the like.

[0208] With flux being conserved, the concentration of detected (in thiscase fluorescent) species changes as a result of a change in velocity.As the label does not change upon conversion of B into B-SA, the numberof labeled molecules in the system remains constant. Where B moleculesare converted to B-SA molecules, taking the principle of theconservation of flux into account:

[B]×U _(B) =[B-SA]×U _(B-SA)

[0209] where [B] is the concentration of B-T10-Fl and U_(B) is thevelocity of the same molecule in the system; U_(B) is relatively slow.[B-SA] is the concentration of the complexed molecule and U_(B-SA) isthe velocity of the complexed molecule, which is relatively fast.Recognition of this relationship allows quantification of the amount ofstreptavidin present in the system by detecting downstream fluorescence.The relationship between the concentrations of B-T10-Fl bound tostreptavidin (B-SA) and unbound to streptavidin (B) is proportional totheir mobilities:

[B-SA]=[B]×U _(B) /U _(B-SA.)

[0210] At intermediate amounts of SA, where a portion of B is bound toSA the concentration is proportional to the fraction (Y_(b)) of B thatis bound to SA:

[Fl]=(1−Y _(b))[B]+Y _(B)([B]U _(B) /U _(B-SA.)

[0211] Without the knowledge that concentration changes as velocitychanges, the assay is much more complicated. For example, one couldsample the mixture into a separation column which separated reacted andunreacted molecules, and detected florescence. The amount of materialcoming off of the column per unit time is optionally detected asdepicted in FIG. 6.

[0212] However, assuming conservation of flux, much simpler arrangementsare possible. For instance, an electrokinetic substrate with one channeland one electrode driving fluid flow in an electrokinetic device isoptionally used to monitor formation of reaction products.

Example 2

[0213] Non-fluorogenic Biotin-streptavidin Binding

[0214] The binding reaction of biotin and streptavidin was chosen as amodel assay to validate the concepts of mobility shift and fluxconservation as a means to detect non-fluorogenic assays in a continuousflow mode. The labeled biotin was a 5′-biotin, 3′-fluoresceinderivatized short oligonucleotide, containing 10 thymidine residues(B-T₁₀-F). The thymidine residues act as a spacer to prevent changes inthe quantum efficiency of fluorescence upon the binding of streptavidinto biotin. Experimentally, it was confirmed by fluorometry (using aPerkin Elmer Luminescence Spectrometer LS50B) that the quantum yield ofB-T₁₀-F was indeed unaffected by the binding reaction to streptavidin.Unlabeled biotin (Sigma B-4501, Lot 37H1389) was also used in this studyas a competitive reactant for BT₁₀-F in the binding reaction withstreptavidin (Sigma S-4762, Lot 44H6890).

[0215] The buffers used for the reagents contain 100 mM Hepes at pH 7.0and 1M NDSB-195 (a non-detergent sulfobetaine, Calbiochem-Novabiochem),filtered with 0.2 μm filters. To vary the electroosmotic mobility of thebuffer solution, a buffer was prepared without added salt and one with50 mM NaCl. A neutral dye, Rhodamine B, was used to measure theelectroosmotic mobility of the buffers.

[0216] All on-chip experiments for this example were performed on aCaliper technologies “7A” chip design; its channel and reagent welllayout is illustrated in FIG. 7. In this design, each reagent well (1,2, and 7) is paired with a buffer well (8, 3, and 6) for on-chipdilution of reagent concentration. The microfluidic channels, 70 μm wideand 10 μm deep, were etched in a soda lime glass substrate and thensealed via thermal bonding with a top glass plate containing eight 3-mmdiameter holes serving as reagent wells. The electrical currents andvoltages of the 8 electrodes in contact with the wells were controlledby a Caliper 3180 LabChip™ controller and Caliper's unified 1 software.

[0217] The fluorescence signals were measured in the epifluorescencemode using a Nikon microscope (Nikon Eclipse TE300) equipped with aphotomultiplier tube (PTI D104 Microscope Photometer) and a 50 Wtungsten/halogen light source. A dichroic filter, High Q FITC Filter Set(#41001, Chroma Technology Corp.), was used for selecting the excitationand emission wavelengths for B-T₁₀-F. A High Q TRITC Filter Set (#41002,Chroma Technology Corp.) was used for Rhodamine B.

[0218] The electroosmotic mobility of the buffers was measured on the 7Achip using Rhodamine B as a neutral dye marker. The electrophoreticmobility of B-T₁₀-F and B-T₁₀-F bound to streptavidin (SA-B-T₁₀-F) wasmeasured directly on the 7A chip using Hepes buffer without NaCl. Forthe μ_(ep) measurements, the concentrations of B-T₁₀-F and SA-B-T₁₀-Fwere 3.1 μm and 0.88 μm, respectively. The measured electrokineticmobilities are tabulated in Table 1. As can be seen from thesemeasurements, B-T₁₀-F has an electrophoretic mobility in the oppositedirection relative to the electroosmotic flow of the buffers due to itsnegative charge at pH 7.0. After the binding reaction, μ_(ep) of theproduct decreases in magnitude due to a decrease in the charge-to-massratio. Thus, the resulting electrokinetic mobility of B-T₁₀-F is lowerthan that of SA-B-T₁₀-F, as in the case described in FIG. 1. TABLE 1Electrokinetic Parameters of Buffers and Reagents Buffer/Reagent μ_(eo)(cm²/v · s) μ_(ep) (cm²/v · s) 100 mM Hepes + 1 M NDSB 5.5 × 10⁻⁴ — 100mM Hepes + 1M NDSB + 50 3.7 × 10⁻⁴ — mMNACL B-T₁₀-F — −2.0 × 10⁻⁴SA-B-T₁₀-F — −0.7 × 10⁻⁴

[0219] A series of experiments was first performed to determine theconcentration of reagents for the binding and competitive binding assayssuch that the signal-to-noise ratio was high and the fluorescence wasstill linear as a function of concentration. The optical setup was alsovaried to ensure that the light intensity and the iris size chosen didnot cause a significant photobleaching of the fluorescent dye.Furthermore, based on model calculations, the buffer with salt givesbetter separation conditions for distinguishing the bound and freeB-T₁₀-F within the geometric and electrical parameters used in ourexperiments on the chip. Therefore, the results reported below wereperformed with the Hepes buffer containing 50 mM NaCl.

[0220]FIG. 8 illustrates the measured fluorescence signal (solid curve)of the non-fluorogenic binding assay of B-T₁₀-F with streptavidin in thecontinuous flow mode. The concentrations of B-T₁₀-F and streptavidinwere 3.1 μm and 78 nM. Since a streptavidin molecule has 4 biotinbinding sites, the stoichiometry of B-T₁₀-F to streptavidin is 10:1. Inthis experiment, the injection time of streptavidin was varied from 2.5s, 5 s, 10 s, and 15 s. The characteristic signature of a peak followed.by a valley can be seen in all cases. For injection times of 10 and 15s, the plateau region is also clearly exhibited. In this plot, the timedomain model calculations using the measured electrokinetic mobilitiesas input parameters are depicted by the dashed curve. It should be notedthat the actual injection pulse shapes were used in the model instead ofan assumed square pulse shape. For quantitative comparison, both themeasured fluorescence and model prediction were first normalized by thebackground fluorescence level when the channel contained only B-T₁₀-F.Furthermore, the magnitude of the model calculations were adjusted byone multiplicative factor to give the best fit to the measured signalsin arbitrary fluorescence units. Thus, the model has one adjustableparameter in the y-axis and no adjustable parameter in the time axis. Ascan be seen in this comparison, the agreement between the model andexperimental data is quite good.

[0221] In another experiment, the competitive binding reaction betweenB-T₁₀-F and unlabeled biotin with streptavidin was studied. FIG. 9 showsthe measured fluorescence signal (solid curve) of the non-fluorogeniccompetitive binding assay results in the continuous flow mode. Thestreptavidin injection time was 12 s. The concentrations of B-T₁₀-F andstreptavidin were 3.1 μm and 78 nM. The concentration of biotin wasvaried at 5 levels: 0, 0.78, 1.6, 2.3, and 3.1 μM. The dashed curve,denoting model calculations based on the actual injection pulseprofiles, was again fitted to the data using one adjustable parameter inthe y-axis as in FIG. 8. As expected, the magnitudes of the peaks andvalleys decrease proportionately as biotin is titrated into the bindingassay to compete with B-T₁₀-F. Once again, the agreement between modelcalculations and measured data is good.

[0222] The data in FIG. 9 is further analyzed by plotting the magnitudeof the peak, plateau, and valley fluorescence level versus thereciprocal of the sum of the labeled and unlabeled biotin concentration.A linear relationship is expected for each set of data for a competitivebinding assay, which was exhibited experimentally as shown in FIG. 10.Any one of these features can be used as a calibration curve todetermine the free biotin concentration in an assay.

[0223] In summary, on-chip data of binding assays of biotin andstreptavidin validated the use of mobility shift to detectnon-fluorogenic assays in a continuous flow mode. The need for productconcentration correction using conservation of flux to analyze assaysperformed in microchannels of a flowing system is also definitivelydemonstrated by a quantitative comparison of data to model calculations.

Example 3

[0224] Applications to Additional Non-Fluorogenic Assays

[0225] The continuous flow, non-fluorogenic assay format can be applieddirectly to binding assays such as of antigen-antibody and receptorligand binding. It is also readily applicable to other biochemicalassays such kinase enzyme assays and hybridization of PNA and acomplimentary peptide nucleic acid (PNA). FIG. 11 shows a plot of somedata on a protein kinase A (PKA) enzyme assay (Promega, V5340) in acontinuous flow mode using a Caliper 7A chip. In this assay, thephosphorylation of the substrate alters the peptide's net charge from +1to −1. Qualitatively, the data (solid curve) shows the expected valleyappearing before the peak, with a plateau region in between. A modelcalculation using estimated electrokinetic mobilities, enzyme kineticparameters from the literature, and estimated applied voltage values inan Excel spatial domain model (dashed curve) predicted the qualitativefeatures of the fluorescent signal. The Macro program listing of thespatial domain model for non-fluorogenic assay is included as AppendixB.

[0226] In the binding assay of biotin and streptavidin presented above,the labeled biotin is a small molecule (244 dalton) whereas theunlabeled streptavidin is large (65,000 dalton). As such, the reactionproduced a labeled product with a significantly different electrokineticmobility compared to the labeled reactant, and this large differencemakes the detection of the binding reaction quite straight forward. Inthe opposite case when the labeled reactant is large (such as a proteinreceptor) and the unlabeled reactant is small (such as a ligand), theinduced mobility shift due to binding could be very small due to a smallchange in the mass. In this case, it is more difficult to detect theonset of reaction using the continuous flow, non-fluorogenic assayformat as described here. However, methods to enhance the detection ofnon-fluorogenic assays on chips for small mobility shifts are availableas described above. One approach is to inject the reaction mixture intoa planar cyclic capillary electrophoresis channel to separate productsfrom reactants. In this case, the separation time can be made very longby continuously cycling the voltage around the cyclic structure. Anothermethod is to use the concept of interference of concentration waves inchannels to enhance to the magnitude of peaks and valleys in thenon-fluorogenic assay fluorescence signal.

Example 4

[0227] High Throughput Systems

[0228] The present invention relates to the performance of assays, andparticularly, high-throughput assays, within microfluidic devices. Theperformance of high-throughput assays within microfluidic devices hasbeen described in great detail in commonly owned published InternationalApplication No. WO 98/00231, as well as supra. Apparatus and methods forintroducing large numbers of different compounds into the microfluidicdevices are described in commonly owned published InternationalApplication No. WO 98/00705, which is also incorporated herein byreference in its entirety for all purposes.

[0229] In many cases, the biochemical system that is being assayed canbe selected or engineered to have an easily detectable result. Forexample, assaying enzyme function is typically made simple by utilizingfluorogenic substrates for the enzyme, e.g., non-fluorescent substrateswhich yield fluorescent products. Such assays are readily incorporatedinto microfluidic devices for performance of assays to identifycompounds that may effect normal enzyme activity. In one embodiment,using a 7A chip as described supra (see, e.g., FIG. 7), one continuouslyflows enzyme and fluorogenic substrate through a channel of the device.This continuous flow of enzyme and substrate produces a steady statefluorescent signal from the fluorescent product. Enzyme inhibitor (or,e.g., compounds' for which one wishes to test inhibitory activity) areperiodically introduced into the main channel. These inhibitors thenreduce the amount of product produced within the main channel resultingin a deviation from the steady state signal. See also, Examples ofspecific assays and their results are shown in the figures attachedherewith. Specifically, both phosphatase assays and protease assays wereperformed using a 7A chip.

[0230] The phosphatase assay utilizes a fluorogenic substrate dFMU,which produces a fluorescent signal upon dephosphorylation. The reactionis shown schematically in FIG. 12. FIG. 13 shows typical data obtainedfrom the on-chip phosphatase assay. In this experiment, the runningbuffer was 1 M NDSB-195 in 25 mM HEPES, pH 7.9. Reagent concentrationswere 125 nM LAR, 50 μM dFMUP and 200 μM peptide inhibitor in wells 6, 8and 2 respectively. Each reagent well was paired with a well containingrunning buffer. The system was programmed to repeatedly run asixteen-step loop of experiments. The sixteen steps were a series ofcontrols followed by the enzyme plus substrate experiment. Each step ofthe loop conserved the total current flux in the main reaction channel.The total flux remained constant during each step of the loop bymaintaining a constant sum of currents from the wells. The proportion ofthat overall flux from each reagent and buffer well was selected toprovide the desired final reagent concentration in the main reactionchannel. The fluorescence response was monitored in each of the sixteenexperimental steps where the continuous flow stream alternated betweenbuffer, substrate, buffer, substrate plus enzyme at four differentsubstrate concentrations. An example of a controller program is shownbelow, Table 2. TABLE 2 Controller Software Program Channel 1 2 3 4 5 67 8 time State: μA μA μA V μA μA μA μA sec 1 5 0 5 1000 0 0 5 0 15Buffer 2 0 0 5 1000 0 0 5 5 15 Substrate 3 5 0 5 1000 0 0 5 0 15 Buffer4 0 0 5 1000 0 5 0 5 15 Substrate + Enzyme

[0231] The substrate concentration was varied for each sequence of threecontrols followed by the enzyme reaction. The concentrations of thereagents in the main channel can be calculated from the ratio ofcurrents used to pump the reagents. The concentrations in the reactionchannel are simply the concentration in the well multiplied by the ratioof current applied at that well, divided by the total current. Here thereaction mixture was 62.5 μM LAR, and either 5, 12.5, 17 or 25 μM dFMUP.The raw fluorescence data is plotted as a function of time. The purposeof this experiment was to demonstrate the increase in enzymatic signalas a function of increasing substrate concentration in a controlledsystem. Rise times for the enzyme/substrate signal are less than 5seconds. The background signal remained low over the course of manyexperimental cycles.

[0232] The raw data for the K_(m), V_(max), k_(cat), and K_(i)determinations are plotted in FIG. 14. Each trace represents a set ofexperiments performed in seven step cycles. The enzyme solution waspumped continuously, providing a final concentration of 83 nM LAR in 1 MNDSB-195, 50 mM HEPES, pH 7.5 in the reaction channel, while the signalat various substrate concentrations was recorded. The first step of thecycle is an enzyme only control. Steps two through six contain differentlevels of substrate up to an including 17 μM dFMUP. The final step ofthe cycle is a substrate only control, 17 μM dFMUP with no enzyme. Theentire experiment, (no peptide inhibitor), was repeated at two inhibitorconcentrations, 35 uM and 69 uM peptide.

[0233] The blank subtracted signals were averaged for triplicatemeasurements and transformed into the reciprocal form of theLineweaver-Burke equation: 1/v=K_(m)/V_(max)×1/[S]+1/V_(max), where v isthe reaction rate in RFU/s, K_(m) is the Michaelis Menton constant forLAR and dFMUP, V_(max) is the rate of maximum enzyme turnover, and S isthe dFMUP concentration. The double reciprocal plot for the range ofsubstrate concentrations, 0-20 μM dFMUP, in the absence of inhibitor,gives K_(m) and V_(max). The rates were evaluated as a change influorescent product signal over a fixed time. The change in fluorescenceis the difference in signal for a given substrate and enzymeconcentration minus the substrate only control. The fixed time is thetime it takes for the product, dFMU, to travel from the point of mixingof substrate and enzyme to the detector. The time for the product toflow was measured directly. dFMU was placed in well 6, the well in whichenzyme typically resides; the time for the product to flow to thedetector poised 8 mm from the source of dFMU in the reaction channel wasmonitored. The slope of a calibration curve of the signal generated as afunction of dFMU concentration was used to convert the fluorescentsignals to dFMU concentrations such that the rates could be expressed asa change in product concentration per unit time.

[0234] A least squares fit of the three straight lines: no inhibitor, 35μM and 69 μM peptide, was performed with the constraint that they meetat a common intercept on the y axis, 1/V_(max) FIG. 15. This fitproduced a V_(max) of 6.71 μM dFMU/s. k_(cat) could then be calculatedfrom the ratio of V_(max) to the enzyme concentration. The k_(cat) is4.74 μmol/min nmol LAR. The parallel analysis performed on thespectrophotometer in the same running buffer yielded a k_(cat) of 6μmol/min nmol LAR. The K_(i) for 35 and 69 μM peptide were 155 and 147μM, respectively. The same analysis performed in a cuvette experimentwith 1 mg/ml BSA in the running buffer gave 167 μM peptide. The data aresummarized in Table 3. TABLE 3 Summary of LAR/dFMUP Kinetic Constants KmKi kcat LAR/dFMUP Peptide μmol/min μM μM mmolLAR Conditions Cuvette 23.2167 6 1 M NDSB-195/50 mM Hepes, pH 7.5, 0.1 mg/ ml BSA Chip 18.7 1514.74 1 M NDSB-195/50 mM Hepes, pH 7.5

[0235] In addition to the above kinetic studies, rate as a function ofsubstrate concentration data was collected on three separate chips inorder to consider interchip reproducibility for K_(m) analyses. Thecombined data were used to prepare a double reciprocal plot. The ratioof the slope to the intercept of the best fit line for these points,(R²=0.999), produced a K_(m) of 18.2 μM. The average of the threeon-chip K_(m) values is 18.7 μM±4.44 (23.8%), n=3. This is in excellentagreement with cuvette experiments performed on the spectrophotometerwhere dFMU was detected at 360 nm in a temperature controlled cuvette at25_C. The cuvette experiments gave an average K_(m) of 23.25 μM±5.25(22.6%), for four separate K_(m) determinations.

[0236] A continuous flow experiment was performed to assess the chiplifetime for the enzyme inhibitor assay. In this experiment 42 nM LARwas continuously pumped through the reaction channel. Alternately, 6.25nM dFMUP or 6.25 nM dFMUP and 41.6 μM peptide inhibitor were pumped intothe flow stream. The reagents were loaded into reagent wells on thechip, the controller was initiated and the script was allowed to run foreight hours. The raw data for the third hour of the experiment is shownin FIG. 16. The entire experiment is summarized by FIG. 17. Note thatalthough both the uninhibited and the inhibited signals drift with time,the percent inhibition remained constant for the entire experiment. Theaverage percent inhibition is 32.45±1.73 (5.3%). From the flow rate andthe cross sectional area of the capillary it is estimated thatapproximately 18 μl total reagent volume was consumed during the eighthours.

[0237] HCV protease was used in a similar fluorogenic assay to LARphosphatase; however, the peptide substrate incorporates a fluorescenceresonance energy transfer (FRET) label (FIG. 18). In order to verifythat the depsipeptide/protease reaction was well behaved and thereaction parameters are in the range we expect, a continuous flow enzymeexperiment with substrate titration was performed. FIG. 19 shows thefluorescence generated in a constant flow stream of 2.14 μM proteasewhen various levels of depsipeptide are introduced, 0 to 250 μMdepsipeptide. The product fluorescence is proportional to the amount ofcleaved substrate. The height of the product signal is proportional tothe rate of enzyme turnover for that substrate concentration. The rateof fluorescence generation can be assessed as the fluorescence signalper mixing time of substrate and enzyme in the reaction channel. Thatmixing time is determined by the mean residence time of the fluorescentproduct in the reaction channel as it is electrokinetically pumped fromthe source of the mixing to the detector. K_(m) was determined from theMichaelis Menton equation.

[0238] Due to the chemistry of this FRET quenching reaction, severalconsiderations for accurate measurement of K_(m) on the Labchip™ exist.(1.) There is not an accurate calibration curve for the EDANS labeledproduct. (2) Accurate determination of the substrate concentration inthe Labchip™ reagent well by a simple spectrophotometric measurement isnot performed. (3) A gross approximation about the fluorescentefficiency of the EDANS-labeled peptide product was made relative toEDANS.

[0239] Despite these considerations, the data from FIG. 19 was convertedto rate information and plotted as a function of the estimateddepsipeptide concentration. The rate values were well behaved and thecorresponding double reciprocal plot is shown in FIG. 20. In aLineweaver-Burke plot, the slope of the line is K_(m)/V_(max), they-intercept is 1/V_(max) and the extrapolated −x intercept is −1/K_(m).Values for K_(m) and k_(cat) derived from a least squares regressionanalysis of the points shown in FIG. 20 are summarized in Table 4 alongwith constants obtained using conventional analysis. TABLE 4Michaelis-Menten Constants Measured on a Labchip ™ and in cuvette forHCV protease and LAR Phosphatase K_(m) V_(max) k_(cat) mM mM/s min⁻¹ HCVProtease/Depsipeptide Kinetics Chip 0.11 0.086 1.8 25 mM TRIS/HCL, pH8.5, 0.1% Triton X-100, 10 mM DTT, 1 M NDSB-195 Cuvette 46 50 mMTRIS/HCL, pH 7.5, 1.0% Triton X-100, 10 mM DTT, 1 *mm EDTA, 10 mM NaClLAR/dFMUP Kinetics chip 0.020-0.40 0.011 3000-5000 50 mM HEPES, pH 7.5,10 mM DTT, 0.5 M NDSB-195

[0240] Note the buffer conditions for the Labchip™ analysis and thetraditional analysis is different. Specifically, pH, surfactantconcentration, and the presence of NDSB are known to influence theenzyme kinetics. Despite this, the agreement between the cuvette valuesand the Labchip™ kinetic constants is reasonable. Moreover, a comparisonof the protease k_(cat) with the phosphatase kinetic constants revealsthe broad range of reaction rates we can expect to accommodate on theLab-chip. It is possible to study the reaction kinetics of enzymes withthree orders of magnitude difference in turnover rate on the sameLabchip™.

[0241] A continuous flow experiment was performed to assess the Labchip™lifetime for the enzyme assay for applications to high throughputscreening. Since the sensitivity of the in vitro enzyme assay is dependson the enzyme concentration employed, in this experiment 0.63 μM HCVprotease was continuously pumped through the reaction channel.Alternately, buffer for 40 seconds or 220 μM depsipeptide for 20 secondswas pumped into the flow stream such that the cycle time for eachexperiment was one minute. Every other substrate injection alsocontained 267 mM inhibitor. The reagents were loaded into reagent wellson the chip, the controller was initiated and the script was allowed torun uninterrupted for more than 12 hours. The raw data for the thirdhour of the experiment is shown in FIG. 21. As expected the inhibitedresponse can be distinguished from the uninhibited substrate generatedsignal, and the peaks are separated by well behaved enzyme only blanks.

[0242] The first 1000 seconds for each hour of the first nine hours ofdata is shown in FIG. 22. The background signal is very stable for thisperiod of time. Note however that this well controlled backgroundfluorescence is not the substrate only background. The extent ofsubstrate hydrolysis over time could not be measured in the continuousflow analysis where enzyme was pumped throughout the course of theexperiments. After nine hours the background increases and the assay nolonger behaves reproducibly. The inhibition reaction is clearly seen forhours one through nine after which time the attenuation of thefluorescence response is not as great. Also the reproducible peak shapesfor hours one through nine start to change after nine hours. The gradualdelay in on time of inhibited and uninhibited peaks for each period ofdata is likely due to deterioration in EO flow. Enzyme adsorbing to thesurface of the capillary can retard the electroosmotic flow, therebyincreasing the incubation time of substrate and enzyme. This producesboth the larger signals and longer mean response times observed here.

[0243] The signals and percent inhibition for hours one through nine aresummarized in FIG. 23. The chip was operational in that fluid wasflowing for more than 12 hours of continuous electrokinetic pumping;however, the inhibition response was reproducible for seven hours. Thetotal reagent volume consumed in the experiment can be calculated fromthe cross sectional area of the capillary and the total current. For a70 mm×20 mm channel and I_(total) equal to 1.5 mA, the reagent volumeconsumed is 2.8 ml/hour or 33 ml in 12 hours. No effort was made tomaximize the number of experiments in this time. Despite this fact,assuming each measurement is an individual experiment, a total of 1680experiments were performed in seven hours. The average percentinhibition response was calculated for the first three inhibited anduninhibited signals at the start of each hour. The percent inhibitionwas 24±2% for the first seven hours of data.

[0244] An example of a non-fluorogenic enzyme assay is depicted in FIG.24. Here a protein kinase reaction is represented in which substrate isconverted to product with differing mobility. Both substrate and productare fluorescently labeled and we rely on the separation of substrate andproduct following conversion to monitor the extent of reaction in a chipdesigned for mixing and incubation followed by separation, e.g. FIG. 25.

[0245] Similar to the phosphatase and protease, the kinase reactivitycan by monitored in the microchip for kinetic analyses and applicationsto high throughput screening. FIG. 26 show the separated peaks due tosubstrate, dye marker, and product as a function of substrateconcentration. The separation occurs following incubation of substrateand enzyme via a gated injection where the flux of substrate and productentering the separation channel is expected to accurately reflect thehomogeneous reaction kinetics. The reaction conditions were 138 nM PKAin 100 mM Hepes, pH 7.5, 10 mM DTT, 5 mM MgCl2, 1M NDSB-195. The doublereciprocal transformation is represented in a Lineweaver Burke plot,FIG. 26 and a Km of 12 uM is derived.

[0246] Non-fluorogenic assays can be designed in various other modes ofoperation. Among the strategies available are assays that modulate theenzyme concentration in a reaction channel containing a constant streamof fluorescent substrate. FIG. 27 contains the trace resulting from aconstant stream of rhodamine-labeled-peptide injected with PKA for 40,30, 30 and 10 second periods. Because the product mobility is fasterthan the substrate mobility under this particular set of conditions, thetrace shows a decrease in substrate concentration due to enzymaticconsumption, followed by an increase in signal of concomitant area dueto an accelerated rate of product generation. Displace substrate isturned over to product and appears as a peak in the fluorescent trace.

[0247] In a similar way utilizing a fluorogenic reaction, here theprotease reaction, constant fluorogenesis can be interrogated withpulses of inhibitor. An example is the protease and peptide substratereaction. This is particularly relevant to high throughput screeningsystems in which continuously flowing enzyme and substrate areelectrokinetically pumped through the reaction channel of the sipperchip and plugs of potential inhibitory compounds are injected. Adecrease in the fluorescence signal should indicate inhibition for thecompounds of interest. In an effort to simulate the high through putexperiment on a planar chip, a constant fluorescence experiment wasconducted. The reaction channel was continuously flowing 1.8 μM HCVProtease and 94 μM depsipeptide. Upon observation of the steady statefluorescence, inhibitor was injected into the flow stream at 75 μM and37.5 μM for 20 s. The total cycle time for injections of twoconcentrations of inhibitor was 240 s. FIG. 28 shows the fluorescencetrace for about 25 minutes.

[0248] Superimposed on the constant fluorescence signal is the inhibitorsignature at two inhibitor concentrations. The higher inhibitorconcentration gives rise to the larger dip followed by a peak. The lowerinhibitor concentration yields a smaller dip followed by a comparablesize peak. The dip and peak pairs are of similar area. We canrationalize these fluorescence responses.

[0249] The depsipeptide has six minus charges while the EDANS labeledproduct contains only two. Therefore we expected, based simply on thedifference in charge, that the substrate should move more slowly in theflow stream than the product. During the time the enzyme “sees”inhibitor in the flow stream, the amount of fluorogenic substrateconsumed is less than that during the uninhibited trace. If the slowmoving substrate lags behind the inhibited response, an increase in theeffective substrate concentration down stream from the inhibition willoccur in the reaction channel. That higher substrate concentration canin turn generate a higher product concentration such that superimposedon the steady state fluorescence signal is a product peak. The similararea of dip and peak for each inhibitor concentration supports thisrationale. The inhibitor concentration dependence of the signatures alsosupports this thinking. In light of the constant fluorescence in theabsence of inhibitor it is likely that a similar experiment may beperformed with shortened inhibitor injection times.

[0250] Modifications can be made to the method and apparatus ashereinbefore described without departing from the spirit or scope of theinvention as claimed, and the invention can be put to a number ofdifferent uses, including:

[0251] The use of an integrated microfluidic system to test the effectof each of a plurality of reaction, assay or components test compoundsin a biochemical or non-biochemical system, the system including datacorrection elements as described herein.

[0252] The use of a microfluidic system as hereinbefore described,wherein said biochemical system flows through one of said channelssubstantially continuously, enabling sequential testing of saidplurality of test compounds, wherein the system includes provisions fordata correction as described.

[0253] The use of a microfluidic system as hereinbefore described,wherein the provision of a plurality of reaction channels in said firstsubstrate enables parallel exposure of a plurality of test compounds toat least one biochemical system, wherein the system includes provisionsfor data correction as described.

[0254] The use of a substrate carrying intersecting channels inscreening test materials for effect on a biochemical system by flowingsaid test materials and biochemical system together using said channelswherein an apparatus utilizing the substrate includes provisions fordata correction as described.

[0255] The use of a microfluidic substrate as hereinbefore described,wherein at least one of said channels has at least one cross-sectionaldimension of range 0.1 to 500 μm.

[0256] The use of a system as described herein for nucleic acidsequencing, wherein the effects of the velocity of labeled components ofa nucleic acid sequencing reaction are corrected for.

[0257] An assay, kit or system utilizing a use of any one of themicrofluidic components, methods or substrates hereinbefore described.Kits will optionally additionally comprise instructions for performingassays or using the devices herein, packaging materials, one or morecontainers which contain assay, device or system components, or thelike.

[0258] In an additional aspect, the present invention provides kitsembodying the methods and apparatus herein. Kits of the inventionoptionally comprise one or more of the following: (1) an apparatus orapparatus component as described herein; (2) instructions for practicingthe methods described herein, and/or for operating the apparatus orapparatus components herein, e.g., for correcting observed concentrationfor effects of velocity; (3) one or more assay component; (4) acontainer for holding apparatus or assay components, and, (5) packagingmaterials.

[0259] In a further aspect, the present invention provides for the useof any apparatus, apparatus component or kit herein, for the practice ofany method or assay herein, and/or for the use of any apparatus or kitto practice any assay or method herein.

[0260] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference for all purposes, as if each reference were specificallyindicated to be incorporated by reference.

What is claimed is:
 1. A method for determining the rate or extent of areaction or assay in a microfluidic system, comprising: converting afirst reaction or assay component having a first velocity (U₁) into aproduct having a second velocity (U_(p)) in a microfluidic channel;determining at least one velocity selected from the group consisting ofU₁ and U_(p); determining the concentration of the reaction or assayproduct in a portion of the microfluidic channel, whereby determiningthe at least one velocity and the concentration of the reaction or assayproduct provides for determination of the rate or extent of the reactionor assay.
 2. The method of claim 1, wherein the first reaction or assaycomponent is converted into the product by exposing the product to heat,light, acid, or base.
 3. The method of claim 1, wherein the firstreaction or assay component is converted into the product by contactingthe first reaction or assay component with a second reaction or assaycomponent.
 4. A method for determining the rate or extent of a reactionor assay in a microfluidic system, comprising: contacting a firstreaction or assay component having a first velocity (U₁) to a secondreaction or assay component having a second velocity (U₂) in amicrofluidic channel, thereby permitting formation of a reaction orassay product with a third velocity (U_(p)); determining at least onevelocity selected from the group consisting of U₁, U₂, and U_(p);determining the concentration of the reaction or assay product in aportion of the microfluidic channel, whereby determining the at leastone velocity and the concentration of the reaction or assay productprovides for determination of the rate or extent of the reaction orassay.
 5. A method for determining the rate or extent of a reaction orassay in an electrokinetic microfluidic system, comprising: providing anelectrokinetic microfluidic device having a microfluidic channel;applying an electric field along the length of the microfluidic channel;contacting a first reaction or assay component having a first chargemass ratio (CM₁) and a first velocity (U₁) to a second reaction or assaycomponent having a second charge mass ratio (CM₂) and a second velocity(U₂) in the microchannel, thereby permitting formation of a reaction orassay product with a third charge mass ratio (CM_(p)) and a thirdvelocity (U_(p)); determining at least one velocity selected from thegroup consisting of U₁, U₂, and U_(p); determining the concentration ofthe reaction or assay product in a portion of the microfluidic channel,whereby determining the at least one velocity and the concentration ofthe reaction or assay product provides for a determination of the rateor extent of the reaction.
 6. The method of claim 5, wherein U₁ isproportional to CM₁, U₂ is proportional to CM₂, and U_(p) isproportional to CM_(P).
 7. The method of claim 1, 4 or 5, the firstreactant or product further comprising a detectable label.
 8. The methodof claim 4 or 5, wherein the velocity of U₁ or U₂ is zero.
 9. The methodof claim 4 or 5, further comprising measuring the velocity of the firstreaction component or the second reaction component and determiningU_(p).
 10. The method of claim 4 or 5, further comprising measuringU_(p).
 11. The method of claim 1, 4 or 5, further comprising determiningthe reaction rate constant (k) for the formation of the product.
 12. Themethod of claim 4 or 5, wherein the second velocity U₂ and the thirdvelocity U_(p) are different.
 13. A method of determining concentrationof a reaction or assay product (C_(p)) in a microfluidic device, themethod comprising the steps of: (i) converting a labeled first reactantor assay component having a velocity (U_(r)) and a label (L_(r)), thelabeled first reactant or assay component producing a signal (S_(as)) ina signal detection system, to a reaction or assay product comprising alabel L_(p), having a velocity (U_(p)), wherein (U_(r)) does not equal(U_(p)) and wherein L_(p) comprises component elements of L_(r); and,(ii) detecting a resulting change in S_(as), wherein the change inS_(as) is an indicator of C_(p).
 14. The method of claim 13, wherein theresulting change in S_(as) is an indicator of U_(r).
 15. The method ofclaim 13, wherein L_(r) comprises a fluorophore.
 16. The method of claim13, wherein the first reactant or assay component is contacted by thesecond reactant or assay component in microfluidic channel in a firstmicrofluidic channel region and wherein S_(as) is detected by monitoringan output from a label detection device which is mounted to view asecond microfluidic channel region in fluid communication with the firstmicrofluidic channel region.
 17. The method of claim 1, 4, 5 or 16,further comprising the step of injecting one or more fluorescent dyes orother flow markers into the microfluidic channel to generate a flowprofile versus time mask file.
 18. The method of claim 1, 4, 5 or 16,further comprising the step of injecting one or more labeled sizemarkers into the microfluidic channel to generate a fluorescenceintensity versus time mask file.
 19. The method of claim 1, 4, 5 or 16,further deconvolution of a complex signal with a time mask file.
 20. Themethod of claim 1, 4, 5 or 16, further comprising baseline subtractionby injecting a series of blanks into the microfluidic channel in acontrol experiment to measure a time dependent baseline.
 21. The methodof claim 1, 4, 5 or 16, further comprising injecting at least one flowmarker into the microfluidic channel, sampling signal from the flowmarker and generating a flow profile versus time mask file.
 22. Themethod of claim 13, further comprising baseline subtraction of reactantsignal (S_(r)) produced by the labeled first reactant from S_(as) toprovide a normalized signal (S_(n)) produced by the product.
 23. Themethod of claim 13, wherein the step of converting the labeled firstreactant or assay component to a reaction or assay product is performedby contacting the labeled first reactant or assay component with asecond reactant or assay component to form a reaction or assay productcomprising a label L_(p) having a velocity (U_(p)), wherein (U_(r)) doesnot equal (U_(p)) and wherein L_(p) comprises component elements ofL_(r).
 24. The method of claim 13 wherein L_(p) is formed from thecomponents of L_(r) by treating the first reactant with a label modifierselected from light, heat, electrical charge, a polymerization agent,and a catalyst.
 25. The method of claim 13, wherein L_(p) and L_(r)comprise the same label moiety.
 26. The method of claim 13, whereinL_(p) and L_(r) comprise different label moieties.
 27. The method ofclaim 4, 5 or 13, wherein the assay or reaction is in a continuous flowformat.
 28. The method of claim 4, 5 or 13, wherein flux is conserved inthe assay or reaction.
 29. The method of claim 4, 5 or 13, wherein thereaction or assay is a non-fluorogenic reaction or assay.
 30. The methodof claim 4, 5 or 13, wherein the first reactant or assay component iscontacted to the second reactant or assay component in a microfluidicchannel.
 31. The method of claim 4, 5 or 13, wherein the first reactantflows down a first channel and the second reactant is periodicallyinjected into the first channel to contact the first reactant.
 32. Themethod of claim 4, 5 or 13, wherein the first reactant or assaycomponent flows down a first microfluidic channel and the secondreactant or assay component is periodically injected into the firstmicrofluidic channel, whereby the first reactant or assay componentcontacts the second reactant or assay component in the firstmicrofluidic channel.
 33. The method of claim 4, 5 or 13, wherein thesecond reactant or assay component is injected into a microfluidicchannel comprising the first reactant for a duration of from 0.001 to 10min.
 34. The method of claim 4, 5 or 13, wherein the second reactant orassay component is reacted with the first reactant or assay component ina non-fluorogenic continuous flow mode.
 35. The method of claim 4, 5 or13, wherein the first reactant or assay component comprises a moietyderived from an antibody, an antigen, a ligand, a receptor, an enzyme,an enzyme substrate, an amino acid, a peptide, a protein, a nucleoside,a nucleotide, a nucleic acid, a fluorophore, a chromophore, biotin,avidin, an organic molecule, a monomer, a polymer, a drug, apolysaccharide, a lipid, a liposome, a micelle, a toxin, a biopolymer, atherapeutically active compound, a I molecule from a biological source,a blood constituent, or a cell.
 36. The method of claim 4, 5 or 13,wherein the first assay component is a component of a biological assay.37. The method of claim 4, 5 or 13, wherein the first assay component isa component of a non-biological assay.
 38. The method of claim 4, 5 or13, wherein the first assay component is a component of a chemicalsynthetic reaction.
 39. The method of claim 4, 5 or 13, furthercomprising contacting the first or second reactant or assay componentwith at least one additional reactant.
 40. The method of claim 4, 5 or13, further comprising the formation of at least one additional reactantor product.
 41. The method of claim 4, 5 or 13, further comprisingdetermining the velocity of an additional reactant, assay component, orproduct.
 42. The method of claim 4, 5 or 13, further comprising the stepof injecting a series of blanks into a channel comprising the firstreactant to determine a time-dependent baseline.
 43. The method of claim4, 5 or 13, wherein the first or second reactant or assay component isdissolved in an aqueous buffer.
 44. The method of claim 4, 5 or 13,wherein the first or second reactant or assay component is dissolved inan aqueous buffer having a pH between 3 and
 11. 45. The method of claim4, 5 or 13, further comprising determining the flux for the firstreaction component, the second reaction component and the product. 46.The method of claim 4, 5 or 13, wherein the first reaction component andthe second reaction component are mixed at a first pH which facilitatesreaction of the first and second reaction component, wherein unreactedfirst component, unreacted second component or product are subsequentlyelectrokinetically transported at a second pH which inhibits reaction ofthe first and second components.
 47. The method of claim 4, 5 or 13,wherein the first reaction component is an enzyme.
 48. The method ofclaim 4, 5 or 13, wherein the first reactant or assay component is anenzyme, the second reactant or assay component is a substrate and theproduct is formed by conversion of the substrate by the enzyme into theproduct.
 49. The method of claim 4, 5 or 13, wherein the first reactantor assay component and the second reactant or assay component hybridizeto form the product, which product has a velocity faster than either thefirst component or the second component.
 50. The method of claim 4, 5 or13, wherein the first reactant or assay component and the secondreactant or assay component hybridize to form the product, which producthas a velocity slower than either the first component or the secondcomponent.
 51. The method of claim 4, 5 or 13, further comprisingmeasuring the concentration of the product spectrophotometrically, oroptically.
 52. The method of claim 4, 5 or 13, wherein the firstreactant or assay component and the second reactant or assay componentcomprise a ligand and a ligand binder, wherein the first componenthybridizes to the second component.
 53. The method of claim 4, 5 or 13,wherein the first reactant or assay component and the second reactant orassay component comprise a ligand and a ligand binder wherein the firstreactant or assay component hybridizes to the second reactant or assaycomponent, and the ligand and ligand binder are selected from the groupconsisting of: a first nucleic acid and a second nucleic acid; anantibody and an antibody ligand; a receptor and a receptor ligand;biotin and avidin; a protein and a complementary protein; and, acarbohydrate and a carbohydrate binding moiety.
 54. The method of claim4, 5 or 13, the first reactant or assay component further comprising abiotin moiety, the second reactant or assay component further comprisinga streptavidin moiety and the product further comprising the biotinmoiety hybridized to the streptavidin moiety.
 55. The method of claim13, wherein the step of converting the labeled first reactant or assaycomponent to a reaction or assay product is performed by contacting thelabeled first reactant or assay component with a second reactant orassay component to form a reaction or assay product comprising a labelL_(p) having a velocity (U_(p)), wherein (U_(r)) does not equal (U_(p))and wherein L_(p) comprises component elements of L_(r), wherein thefirst reactant or assay component and the second reactant or assaycomponent are contacted in a microfluidic channel.
 56. The method ofclaim 4, 5 or 55, further comprising measuring the concentration of theproduct in a microfluidic channel, and, optionally, measuring theconcentration of the first reaction or assay component in a portion ofthe microfluidic channel and, optionally, measuring the concentration ofthe second reaction or assay component in a portion of the microfluidicchannel.
 57. The method of claim 4, 5 or 55, further comprisingmeasuring a length of time for travel of the first reaction component orthe second reaction component along a selected length of themicrofluidic channel.
 58. The method of claim 4, 5 or 55, furthercomprising measuring a length of time for travel of the product along aselected length of the microfluidic channel.
 59. The method of claim 4,5 or 55, wherein the first component, the second component, and theproduct are soluble in an aqueous solvent, wherein the microchannelcomprises said aqueous solvent.
 60. The method of claim 4, 5 or 55,further comprising providing an electrokinetic microfluidic devicehaving the microfluidic channel; and, applying an electric field alongthe length of the microchannel.
 61. The method of claim 4, 5 or 55,wherein the first and second component have a K_(a) of between about 10⁵and 10¹⁵.
 62. A method of detecting a product formed by contacting afirst and second component of a reaction comprising: contacting thefirst and second reactant in a microfluidic channel, wherein the firstreactant comprises a detectable label, thereby producing a productcomprising 5 the detectable label, which product has a differentelectrokinetic mobility than the first or second reactant; flowing theproduct and any first or second reactant remaining in the channelsubsequent to said contacting step past a detector, wherein the label onthe first reactant and the label on the product comprise the samedetectable moiety; and, determining at least one of: concentration ofthe product, rate of product formation, or amount of product produced.63. The method of claim 62, wherein the detectable label is afluorophore.
 64. The method of claim 62, wherein the detectable label isa fluorophore and the reaction is non-fluorogenic.
 65. The method ofclaim 62, the method further comprising measuring or calculating thevelocity of the first reactant, the second reactant, or the product. 66.The method of claim 62, wherein flux of the detectable label isconserved.
 67. The method of claim 62, wherein the first or secondreactant is periodically injected into the channel.
 68. The method ofclaim 62, wherein the first reactant, the second reactant and theproduct are flowed continuously in the channel.
 69. The method of claim62 further comprising detecting phase shift of reactant and productwaves.
 70. A method of dispensing representative mixtures in amicrofluidic system, comprising: (i) introducing a first mixture into afirst microfluidic channel, the mixture comprising at least first andsecond materials; (iii) transporting the first and second materialsthrough the first channel, wherein the first and second mixtures travelat different velocities in the channel; (iv) gating an aliquot of firstand second materials into the second channel for a selected period oftime, the relative amount of first and second materials in the aliquotbeing proportional to the flux of first and second materials in thefirst mixture in the first channel, thereby dispensing a representativemixture of the first and second components.
 71. The method of claim 70,wherein flux is conserved in the system.
 72. The method of claim 70,wherein the first and second compounds have different fluxes duringelectrokinetic movement.
 73. The method of claim 70, wherein the firstor second material is labeled, the method comprising measuring signalfrom the aliquot of first or second labeled material, wherein the amountof labeled material is determined by measuring the signal.
 74. Themethod of claim 70, comprising providing a microfluidic devicecomprising a body structure having at the first channel and at least asecond channel disposed therein, the first and second channelscommunicating at a first intersection.
 75. The method of claim 74,wherein the first and second channels communicate at a crossingintersection.
 76. The method of claim 70, wherein the first and secondmaterials are moved electrokinetically in the first channel.
 77. Themethod of claim 70, further comprising measuring the amount of first orsecond material in the aliquot.
 78. The method of claim 70, wherein thefirst material is a reactant and the second material is a product of areaction of the reactant.
 79. The method of claim 70, wherein aseparation of the first and second materials occurs in the secondchannel within the aliquot.
 80. The method of claim 70, wherein thealiquot is injected into the second channel by a voltage change.
 81. Themethod of claim 70, wherein the aliquot is injected into the secondchannel by a current change.
 82. The method of claim 70, the methodfurther comprising detecting the first or second material using a totalamount detector which measures label across the entire aliquot.
 83. Themethod of claim 70, the method further comprising detecting the first orsecond material with a label detector comprising a wide photomultipliertube slit and a photomultiplier tube.
 84. The method of claim 70, themethod further comprising detecting the first or second material bytotal photobleaching, a long window fluorescent detector or anelectrochemical detector which samples the entire aliquot.
 85. A methodof correcting data in a microfluidic system for effects of stacking ofcharged molecules in a microfluidic channel comprising: injecting atleast one labeled blank into the microfluidic channel; monitoringcontrol signal from the labeled blank in the channel to determine thesignal of the blank over time; and, subtracting the control signal ofthe blank over time from experimental data from an analyte in themicrofluidic channel.
 86. The method of claims 85, further comprisinginjecting at least one flow marker into the microfluidic channel,sampling signal from the flow marker and generating a flow profileversus time mask file.
 87. A method of correcting data in a microfluidicsystem for effects of stacking of charged molecules in a microfluidicchannel comprising: injecting a series of labeled control molecules indiscreet high-salt buffer control plugs into the microfluidic channel tocharacterize timing of the control plugs as they pass the detectionpoint; creating a control label intensity versus time data mask file;and, correlating the label intensity versus time mask file toexperimental data from an analyte in the microfluidic channel todetermine which times from the experimental data correlate with a sampleplug.
 88. The method of claims 85 or 87 wherein the label isfluorescent.
 89. A method of regulating a flowing reaction in amicrofluidic channel comprising: mixing a plurality of reactioncomponents in a first buffer, thereby providing a mixture of reactioncomponents; electrokinetically transporting the mixture of reactioncomponents in a microfluidic channel, thereby permitting the mixture ofreaction components to react; applying a reaction inhibitor to at leasta portion of the reaction mixture, thereby inhibiting further reactionof the reaction components in the portion.
 90. The method of claim 89,wherein the inhibitor is selected from: an aliquot of high pH buffer; analiquot of low pH buffer; an aliquot of buffer comprising an ionchelator; an aliquot of high temperature buffer, an aliquot of lowtemperature buffer, heat, and light.
 91. The method of claim 89, whereinthe inhibitor is applied to selected regions of the flowing mixture ofreaction components, wherein the selected regions bracket regions whichare not selected in which the inhibitor is not applied.
 92. The methodof claim 89, wherein the inhibitor is added in a time-gated aliquot. 93.The method of claim 89, wherein the mixture of reaction components areelectrokinetically flowed for a selected period of time.
 94. The methodof claim 89, wherein the mixture of reaction components areelectrokinetically flowed for a selected distance in the microfluidicchannel.
 95. A microfluidic apparatus for determining a rate offormation of a moving analyte on an electrokinetic microfluidicsubstrate comprising: a microfluidic substrate holder for receiving amicrofluidic substrate during operation of the apparatus, whichsubstrate holder has a microfluidic substrate viewing region; an analytedetector mounted proximal to the substrate viewing region to detect themoving analyte in a portion of the substrate viewing region; and, acomputer operably linked to the analyte detector, which computerdetermines the rate of formation of the analyte, correcting for theeffects of the motion of the analyte.
 96. A microfluidic apparatus fordetermining a rate of formation of a moving analyte on an electrokineticmicrofluidic substrate comprising: a microfluidic substrate holder forreceiving a microfluidic substrate during operation of the apparatus,which substrate holder has a microfluidic substrate viewing region;analyte movement means for imparting velocity to the analyte in achannel of the microfluidic substrate during operation of the apparatus;detection means for detecting the moving analyte in the substrateviewing region; and, correction means for correcting the observed rateof formation of the moving analyte for the effects of the velocity ofthe analyte, which means are operably linked to the means for detectingthe moving analyte.
 97. The microfluidic apparatus of claim 96, whereinthe correction means comprise a computer operably linked to thedetection means, which computer determines the rate of formation of theanalyte, and which computer corrects for the effects of the motion ofthe analyte.
 98. The microfluidic apparatus of claim 95, 96, wherein theapparatus is in use and further comprises a microfluidic substratemounted in the microfluidic substrate holder.
 99. A microfluidicapparatus, comprising: a microfluidic substrate comprising a body havinga top portion, a bottom portion and an interior portion; the interiorportion comprising at least two intersecting channels, wherein at leastone of the two intersecting channels has at least one cross sectionaldimension between about 0.1 μm and 500 μm; a detection zone fordetecting the analyte in at least one of the two intersecting channels,when the analyte is in motion; and, a data analyzer which determines arate of formation of the analyte in motion, wherein the analyzercomprises a processor which calculates the flux or velocity of theanalyte in the detection zone.
 100. The microfluidic apparatus of claim99, wherein the apparatus is formed by etching at least two intersectinggroves in a top surface of the bottom portion, the top portion beingfused to the top surface of the bottom portion, thereby forming theinterior portion.
 101. The microfluidic apparatus of claim 99, the dataanalyzer comprising a computer with software for determining the rate offormation of moving analytes on a microfluidic device in which flux isconserved.
 102. The microfluidic apparatus of claim 99, the top portionof the device further comprising a plurality of wells in fluidcommunication, and an electrokinetic fluid direction system comprising aplurality-of electrodes adapted to fit into the plurality of wells. 103.The microfluidic apparatus of claim 95, 96 or 99, wherein the apparatuscomprises an optical or fluorescent detection system for viewing theanalyte.
 104. The apparatus of claim 95, 96 or 99 comprising anelectrokinetic fluid direction system.
 105. The apparatus of claim 104,comprising an electrode disposed within a well formed in the top portionof the body.
 106. The apparatus of claim 95, 96 or 99 further comprisinga microscope.
 107. An apparatus for determining the concentration of aproduct in a non-fluorogenic format, comprising: conversion means forconverting a labeled first reactant or assay component to a secondlabeled component; signal detection means for detecting signal amplitudefrom the labeled first reactant or assay component and second labeledcomponent; concentration calculation means for calculating theconcentration of the product by measuring a change in signal amplitudewhich results from converting the first reactant or assay component intothe second labeled component.
 108. The apparatus of claim 107, whereinthe signal detection means comprises an optical detector for detecting alight signal.
 109. The apparatus of claim 107, wherein the signaldetection means comprises an optical detector for detecting afluorescent signal.
 110. The apparatus of claim 107, wherein theconcentration calculation means comprises a digital computer.
 111. Theapparatus of claim 107, wherein the conversion means comprises amicrofluidic substrate having at least two intersecting channelsfabricated therein.
 112. An apparatus for determining the concentrationof a product in a non-fluorogenic format, comprising: a microfluidicsubstrate holder for receiving a microfluidic substrate, the holdercomprising a substrate viewing region; a signal detector mountedproximal to the substrate viewing region; a signal output processorwhich converts variations in signal amplitude from the signal detectorinto concentration measurements for at least one of a plurality ofmoving analytes comprising one or more labels which have the same signaloutput, which plurality of analytes are detected by the signal detector.113. The apparatus of claim 112, wherein the signal detector detects oneor more label selected from the group of a fluorescent label, acalorimetric label, and, a radioactive label.
 114. The apparatus ofclaim 112, comprising: a microfluidic substrate having a plurality ofmicrochannels fabricated therein, the substrate mounted in the substrateholder, wherein the apparatus in use comprises a first analytecomprising a first label, and a second analyte comprising the same firstlabel, wherein the mobility of the first and second analyte aredifferent.
 115. The apparatus of claim 107 or 112, further comprising anelectrokinetic fluid control means.
 116. The apparatus of claim 107 or112, comprising a microfluidic substrate with a reaction channelfabricated therein, wherein, during use of the apparatus, first andsecond reactants are contacted in the reaction channel, wherein thereaction channel is in fluid communication with a first reagentintroduction channel, wherein the second reactant is introduced into thereaction channel from the first reagent introduction channel by timegated injection.